Nucleic acid transporter systems and methods of use

ABSTRACT

Nucleic acid transporter systems for delivery of nucleic acid to a cell. The nucleic acid transporter includes a binding complex. The binding complex contains a binding molecule which non-covalently binds to the nucleic acid and covalently links to a surface ligand, nuclear ligand and/or a lysis agent. These may be linked to the binding molecule by spacers.

The invention was partially supported by a grant from the United States government under H.L.-23741 awarded by the National Institutes of Health. The U.S. government may have rights in the invention.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/167,641, by Woo et al., filed Dec. 14, 1993, now U.S. Pat. No. 6,033,884, entitled "Nucleic Acid Transporter Systems and Methods of Use". The Ser. No. 08/167,641 application is a continuation-in-part of Woo et al., application Ser. No. 07/855,389, filed Mar. 20, 1992, now abandoned, entitled "A DNA Transporter System and Method of Use," the whole of which (including drawings) is hereby incorporated by reference. This application is also a continuation-in-part of Woo et al., International Application No. PCT/US93/02725, filed Mar. 19, 1993 (designating U.S. and other countries), entitled "A DNA Transporter System and Method of Use", the whole of which (including drawings) is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

This invention relates to gene therapy using transporter systems for delivering nucleic acid into a cell.

Recombinant retroviral vectors have been used for delivery of genes to cells of living animals. Retroviral vectors permanently integrate the transfered gene into the host chromosal DNA. Studies have demonstrated that retroviral vectors can be transduced into liver cells in vivo by direct injection of the virus into the parenchyma after carbon tetrachloride treatment. Kaleko et al., Human Gene Therapy, Vol. 2, pp. 27-32 (1991). Analysis of transduced liver cells indicated a transduction frequency of about 1 out of 160 liver cells. Id.

Retroviral vectors can be transduced into rat hepatocytes after partial hepatectomy followed by a surgical procedure involving isolation and perfusion of the liver with a viral vector. Poorman et al., Arterioscl. Throm., Vol. 11, p. 1413A (1991); Hobbs et al., Annu. Rev. Genet., Vol. 24, pp. 133-70 (1990). The transduced hepatocytes from the above studies were detectable six months after gene transduction. Id.

Another virus used for gene delivery is adenovirus. It has been developed as a means for gene transfer into epithelial derived tissues. Stratford-Perricaudet et al., Hum. Gene. Ther., Vol. 1, pp. 241-256 (1990); Gilardi et al., FEBS, Vol. 267, pp. 60-62 (1990); Rosenfeld et al., Science, Vol. 252, pp. 431-434 (1991). Vectors have been constructed in which the 35 Kb genome in the E3 and E1 regions have been deleted, such that recombinant gene constructs can be inserted into the adenovirus vector. Id. Since adenovirus has a natural tropism for the lung epithelium it was used for gene transfer into this tissue. Gilardi et al., FEBS, Vol. 267, pp. 60-62 (1990); Rosenfeld et al., Cell, Vol. 68, pp. 143-155 (1992).

Recombinant adenoviral vectors have the advantage over retroviruses of being able to transduce non-poliferating cells as well as an ability to produce purified high titer virus. Studies using an adenoviral vector to deliver genes to liver demonstrated that mouse hepatocytes can be transduced in vivo with the vector. Stratford-Perricaudet, Hum. Gene Ther., Vol. 1, pp. 241-256 (1990). Use of adenoviral-mediated transfer of orninthine transcarbamylase cDNA allowed the transfer of enzyme activity to the mouse liver. These studies resulted in phenotypic correction of enzyme deficiency. Id. Other studies have demonstrated human α-1-antitrypsin production from rat liver after transduction with a recombinant adenoviral vector. Jaffe et al., Nature-Genetics, Vol. 1, pp. 372-378 (1992). These studies determined that 1% of hepatocytes were transduced in vivo. Id.

In addition to retroviral-mediated gene delivery, a more recent means for DNA delivery has been receptor-mediated endocytosis. Endocytosis is the process by which eucaryotic cells continually ingest segments of the plasma membrane in the form of small endocytotic vesicles. Alberts et al., Mol. Biol. of Cell, Garland Publishing Co., New York, 1983. Extracellular fluid and everything dissolved in it becomes trapped in the vesicle and is ingested into the cell. Id. This process of bulk fluid-phase endocytosis can be visualized and quantified using a tracer such as enzyme peroxidase introduced into the extra-cellular fluid. Id. The rate of constitutive endocytosis varies from cell type to cell type.

Endocytotic vesicles form in a variety of sizes and shapes and are usually enlarged by fusing with each other and/or with other intra-cellular vesicles. Stryer, Bioch., Freeman and Co., New York (1988). In most cells the great majority of endocytotic vesicles ultimately fuse with small vesicles called primary lysosomes to form secondary lysosomes which are specialized sites of intra-cellular digestion. Id. The lysosomes contain a wide variety of degradative enzymes to digest the macromolecular contents of the vesicles. Silverstein, et al., Ann. Rev. Biochem., Vol. 46, pp. 669-722 (1977); Simionescu, et al., J. Cell Biol., Vol. 64, pp. 586-607 (1975).

Many of the endocytotic vesicles are coated and are formed by invagination of coated regions of the plasma membrane called coated pits. Coated pits and vesicles provide a specialized pathway for taking up specific macromolecules from the extracellular fluid. This process is called receptor-mediated endocytosis. Goldstein et al., Nature, Vol. 279, pp. 679-685 (1979); Pearse, et al., Ann. Rev. Biochem., Vol. 50, pp. 85-101 (1981); Postan, et al., Ann. Rev. Physiol., Vol. 43, pp. 239-250 (1981). The macromolecules that bind to specific cell surface receptors are internalized via coated pits. Goldstein, supra. Receptor-mediated endocytosis is a selective mechanism enabling cells to ingest large amounts of specific ligands without taking in correspondingly large amounts of extra-cellular fluid. Goldstein, supra.

One such macromolecule is low density lipoprotein ("LDL"). Numerous studies have been performed involving LDL and the receptor-mediated endocytotic pathway. In addition to LDL, many other cell surface receptors have been discovered to be associated with coated pits and receptor-mediated endocytosis. Pastan et al., Ann. Rev. Physiol., Vol. 43, pp. 239-250 (1981). For example, studies have analyzed the hormone insulin binding to cell surface receptors and entering the cell via coated pits. Stryer et al., Biochemistry, Freeman & Co., New York (1988); Alberts et al., Molecular Biology of the Cell, Garland Publishing, New York (1983). In addition, it has been determined that some cell surface receptors associate with coated pits only after ligand binding. Pastan, supra.

Taking advantage of receptor-mediated endocytosis, the asialoglycoprotein receptor has been used in targeting DNA to HepG2 cells in vitro and liver cells in vivo. Wu and Wu, J. Biol. Chem., Vol. 262, pp. 4429-4432 (1987); Wu and Wu, Bio., Vol. 27, pp. 887-892 (1988); Wu et al., J. Biol. Chem., Vol. 263, pp. 14621-14624 (1988); Wu et al., J. Biol. Chem., Vol. 264, pp. 16985-16987 (1989); Wu et al., J. Biol. Chem., Vol. 266, pp. 14338-14342 (1991). These studies used asialoorosomucoid covalently linked to polylysine with water soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or with 3'(2'pyridyldithio)propionic acid n-hydroxysuccinimide ester. Polylysine in the studies above bound DNA through ionic interaction. The DNA was ingested by endocytosis.

Other studies have utilized transferrin and the transferrin receptor for delivery of DNA to cells in vitro. Wagner et al., P.N.A.S., Vol. 87, pp. 3410-3414 (1990). These studies modified transferrin by covalently coupling transferrin to polylysine. Id. The polylysine interacted ionically with DNA. Delivery of DNA occurred to cells through the transferrin receptor. Such analyses were performed in vitro. Id. Cotten et al., P.N.A.S., Vol. 87, pp. 4033-4037 (1990); Zenke et al., P.N.A.S., Vol. 87, pp. 3655-3659 (1990).

In addition to DNA, macromolecules can also be delivered by receptor-ligand systems. Leamon et al., P.N.A.S., Vol. 88, pp. 5572-5576 (1991); Leamon et al., J. Biol. Chem., Vol. 267, pp. 24966-24971 (1992). In particular these studies have involved the folate receptor, an anchored glycosyl-phosphatidyl protein, which is excluded from coated pits and cycles in and out of the cells by caveolae. Hill and Dessler, Science, Vol. 252, pp. 410-414 (1991). This uptake mechanism has been called potocytosis. Id. Folate conjugated enzymes have been delivered into cells through this receptor system and retained activity for at least 6 hours. Leamon et al., P.N.A.S., Vol. 88, pp. 5572-5576 (1991). Folate receptors have limited tissue distribution and are overexpressed in several malignant cell lines derived from many tissues. Weitman et al., Cancer Res., Vol. 52, pp. 3396-3401 (1992); Weitman et al., Cancer Res., Vol. 52, pp. 6708-6711 (1992); Campbell, Cancer Res., Vol. 51, pp. 5329-5338 (1991); Coney, Cancer Res., Vol. 51, pp. 6125-6132 (1991). Other studies have also used biotin or folate conjugated to proteins by biotinylation for protein delivery to the cell. Low et al., U.S. Pat. No. 5,108,921.

DNA and macromolecule delivery is hindered by lysosomal degradation. Studies have analyzed the endosomal/lysosomal degradation process. It has been determined that organisms which are internalized via receptor-mediated endocytosis or receptor:ligand systems, like viruses and other microorganisms, escape lysosomal degradation in order to function. The entry mechanism of some viruses have been studies extensively. For some viruses outer membrane proteins have been demonstrated to be important for endosomal escape. Marsh et al., Adv. Virus Res., Vol. 36, pp. 107-151 (1989). Other studies have focused on methods to prevent lysosomal degradation. These studies have used substances which pertubate endosomal/lysosomal function. Mellmann et al., Ann. Rev. Biochem., Vol. 55, pp. 663-700 (1986). These substances have only been used in vitro. In addition, studies show that the entire virus-shell is necessary for efficient endosomal lysis. Marsh et al., Adv. Virus Res., Vol. 36, pp. 107-151 (1989). Studies have also demonstrated that adenovirus will enhance transferrin-polylysine mediated gene delivery. Curiel P.N.A.S., Vol. 88, pp. 8850-8854 (1991).

A number of bacteria are also internalized via receptor-mediated endocytosis and are liberated from the endosome by production of toxins. These toxins lyse the endosomal membrane. Moulder, Microbiol. Rev., Vol. 49, pp. 298-337 (1985). Listeria monocytogenes produce a membranolytic toxin called listeriolysin. Cossart et al., Mol. Biol. Med., Vol. 6, pp. 463-474 (1989); Tilney et al., J. Cell Bio., Vol. 109 pp. 1597-1608 (1989). Studies have shown that no other cofactors are needed for endosomal escape of Listeria monocytogenes. Bielecki et al., Nature Vol. 345 pp. 175-176 (1990).

The listeriolysin toxin forms pores in membranes which contain cholesterol. These pores are large enough for macromolecules like immunoglobulins to pass. Ahnert-Hilger et al., Mol. Cell Biol., Vol. 31 pp. 63-90 (1989); Geoffroy et al., J. of Bacteriol., Vol. 172, pp. 7301-7305 (1990).

SUMMARY OF THE INVENTION

Applicant has determined that it is useful to construct nucleic acid transporter systems for delivering nucleic acid into the cell. Specifically, these transporter systems deliver nucleic acid into the cellular interior as well as the nucleus. These are useful in targeting nucleic acid to specific cells. These transporters can be used to treat diseases by targeting specific nucleic acid accordingly. These transporters can also be used to create transgenic animals for assessing human disease in an animal model.

The present invention takes advantage of the unique targeting ability to deliver nucleic acid to specific cells, the unique ability to release nucleic acid into the cellular interior and the unique ability to direct nucleic acid into the nucleus of a cell. The present invention features use of nucleic acid binding complexes containing surface ligands which are capable of binding to a cell surface receptor and entering a cell through cytosis (e.g., endocytosis, potocytosis, pinocytosis). In particular, the present invention demonstrates that by using surface ligands specific to certain cells, nucleic acid can be delivered using the nucleic acid transporter systems directly to the desired tissue. In addition, the present invention features use of a nucleic acid binding complex with a nuclear ligand capable of recognizing and transporting nucleic acid through the nuclear membrane to the nucleus of a cell.

Furthermore, to avoid the problems of endosomal/lysosomal degradation, the present invention takes advantage of lysis agents. In particular, the present invention features use of a nucleic acid binding complex which includes a lysis agent capable of releasing nucleic acid into the cellular interior from the endosome. The nucleic acid can be efficiently released without endosomal/lysosomal degradation.

The unique targeting ability to specific cells and to the nucleus also allows transgenic animal models to be used for the dissection of molecular carcinogenesis and disease, assessing potential chemical and physical carcinogens and tumor promoters, exploring model therapeutic avenues as well as livestock agricultural purposes. Furthermore, the above nucleic acid transporter system advantages allow methods for administration and treatment of various diseases. In addition, the above nucleic acid transporter systems can be used to transform cells to produce particular proteins, polypeptides, and/or RNA. Likewise, the above nucleic acid transporter systems can be used in vitro with tissue culture cells. In vitro uses allow the role of various nucleic acids to be studied by targeting specific expression into specifically targeted tissue culture cells.

In the first aspect, the present invention features a nucleic acid transporter system for delivering nucleic acid into a cell. The nucleic acid transporter includes a nucleic acid binding complex. This nucleic acid binding complex includes a binding molecule which is noncovalently bound to nucleic acid. In addition, the binding molecule is covalently linked to a surface ligand.

The term "nucleic acid transporter system" as used herein refers to a molecular complex which is capable of efficiently transporting nucleic acid through the cell membrane. This molecular complex is bound to nucleic acid noncovalently. In addition to nucleic acid, other macromolecules including but not limited to, proteins, lipids and carbohydrates can also be delivered using the transporter system. The nucleic acid transporter system is capable of releasing the noncovalently bound nucleic acid into the cellular interior. Although not necessary, the nucleic acid transporter system can also efficiently transport the nucleic acid through the nuclear membrane. Furthermore, the nucleic acid transporter may also prevent degradation of the nucleic acid by endosomal lysis.

The nucleic acid transporter system as described herein can contain but is not limited to five components. It comprises, consists or consists essentially of: (1) a nucleic acid or other macromolecule with a known primary sequence that contains the genetic information of interest or a known chemical composition; (2) a moiety that recognizes and binds to a cell surface receptor or antigen or is capable of entering a cell through cytosis; (3) a nucleic acid or macromolecular molecule binding moiety; (4) a moiety that is capable of moving or initiating movement through a nuclear membrane; and/or (5) a lysis moiety that enables the transport of the entire complex from the cell surface directly into the cytoplasm of the cell. The term "consisting of" is used herein as it is recognized in the art. The transporter "consisting essentially of" the five moieties above includes variation of the above moieties. Such a variation may be use of only three moieties instead of all five. This is only an example and is non-limiting.

The term "nucleic acid" as used herein refers to DNA or RNA. This would include naked DNA, a nucleic acid cassette, naked RNA, or nucleic acid contained in vectors or viruses. These are only examples and are not meant to be limiting.

The term "vector" as used herein refers to nucleic acid, e.g., DNA derived from a plasmid, cosmid, plasmid or bacteriophage into which fragments of nucleic acid may be inserted or cloned. The vector can contain one or more unique restriction sites for this purpose, and may be capable of autonomous replication in a defined host or organism such that the cloned sequence is reproduced. The vector molecule can confer some well-defined phenotype on the host organism which is either selectable or readily detected. Some components of a vector may be a DNA molecule incorporating DNA, a sequence encoding a therapeutic or desired product, and regulatory elements for transcription, translation, RNA stability and replication. A viral vector in this sense is one that contains a portion of a viral genome, e.g. a packaging signal, and is not merely DNA or a located gene within a viral particle.

Expression includes the efficient transcription of an inserted gene or nucleic acid sequence within the vector. Expression products may be proteins, polypeptides or RNA. The gene insert or nucleic acid sequence may be contained in a nucleic acid cassette.

The term "nucleic acid cassette" as used herein refers to the genetic material of interest which can express a protein, polypeptide or RNA. The nucleic acid cassette can be naked DNA or positionally and sequentially oriented within a vector such that the nucleic acid in the cassette can be expressed, i.e., transcribed into RNA, and when necessary, translated into a protein or a polypeptide.

A variety of proteins and polypeptides can be encoded by the sequence in a nucleic acid cassette. Those proteins or polypeptides which can be expressed include hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, viral antigens, parasitic antigens and bacterial antigens. Specific examples of these compounds include proinsulin, insulin, growth hormone, androgen receptors, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding proteins, epidermal growth factor, TGF-α, TGF-β, dermal growth factor (PDGF), angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor and angiogenin), matrix proteins (Type IV collagen, Type VII collagen, laminin), oncogenes (ras, fos, myc, erb, src, sis, jun), E6 or E7 transforming sequence, p53 protein, cytokine receptor, IL-1, IL-6, IL-8, viral capsid protein, and proteins from viral, bacterial and parasitic organisms. Other specific proteins or polypeptides which can be expressed include: phenylalanine hydroxylase, α-1-antitrypsin, cholesterol-7α-hydroxylase, truncated apolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoprotein A1, LDL receptor, molecular variants of each, and combinations thereof. One skilled in the art readily appreciates that these proteins belong to a wide variety of classes of proteins, and that other proteins within these classes can also be used. In addition, the nucleic acid cassette can code for antisense RNA or ribozymes as well. These are only examples and are not meant to be limiting in any way.

The term "nucleic acid binding complex" as used herein refers to a complex which includes a binding molecule. The binding molecule is capable of noncovalently binding to nucleic acid. The binding molecule is also capable of covalently linking to a surface ligand, a nuclear ligand and/or a lysis agent. The binding molecule can include but is not limited to spermine, spermine derivative, spermidine, histones, polylysine, polyamines and cationic peptides. In addition, this includes but is not limited to analogs or derivatives of the above compounds.

The term "spermine" refers to a cation capable of noncovalent binding with nucleic acid through electrostatic components. Such binding can include ionic interaction, hydrogen bonding, and hydrophobic bonding. The term "derivative" as used herein refers to a compound produced from another compound of similar structure in one or more steps. For example, this includes spermine analogs and any chemical variation of spermine. This would also include any change in the structure of spermine but with the desired activity still remaining. Such a change, for example, could be a change in a chemical bond, or a change in a hydrogen placement. This is only an example and is nonlimiting. In addition, "analog" as used herein refers to a compound that resembles another structure, e.g., spermine, but is not necessarily an isomer.

Spermine derivatives include compounds IV, VII, XXI, XXXIII, XXXVI, LIV, LVI, LXXXII, LXXXIV and CX as described below. When used with the nucleic acid transporter system, the binding molecules, whether attached to a surface ligand, nuclear ligand or a lysis agent, can be different or similar binding molecules. In a preferred embodiment the binding molecule is a spermine derivative labeled below as D, as shown in FIG. 18.

Spermine and spermine derivatives have advantages over poly-1-lysine as used for the binding moledule. The binding properties of the spermine derivatives will approximate most closely those of spermine. The intranuclear spermine concentration is approximately 3 to 10 mmol. Spermine and spermine derivatives of this present invention are advantageous to use for two main reasons. First, the spacing of the amino groups of spermine is such that this naturally occuring polycation fits into the major groove of the DNA double helix with an exact fit. While the polycationic polylysine interacts electrostatically with the phosphates in the groove of DNA, the fit is not as precise. Second, the theoretical association/disassociation kinetics of the DNA/spermine interaction are more rapid for the DNA/spermine interactions than for DNA/polylysine. This is advantageous in the spermine/DNA mix for the release of the DNA inside the cell.

The term "surface ligand" as used herein refers to a chemical compound or structure which will bind to a surface receptor of a cell. The term "cell surface receptor" as used herein refers to a specific chemical grouping on the surface of a cell for which the ligand can attach. Cell surface receptors can be specific for a particular cell, i.e., found predominantly in one cell rather than in another type of cell (e.g., LDL and asialoglycoprotein receptors are specific for hepatocytes). The receptor facilitates the internalization of the ligand and attached molecules. A cell surface receptor includes but is not limited to a folate receptor, biotin receptor, lipoic acid receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, insulin-like growth factor type II/cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, cartilage induction factor receptor or glycosylphosphatidylinositol (GPI)-anchored proteins (e.g., β andrenargic receptor, T-cell activating protein, Thy-1 protein, GPI-anchored 5' nucleotidase). These are nonlimiting examples.

A receptor is a molecule to which a ligand binds specifically and with relatively high affinity. It is usually a protein or a glycoprotein, but may also be a glycolipid, a lipidpolysaccharide, a glycosaminoglycan or a glycocalyx. For purposes of this invention, epitopes to which an antibody or its fragments binds is construed as a receptor since the antigen:antibody complex undergoes endocytosis. Furthermore, surface ligand includes anything which is capable of entering the cell through cytosis (e.g. endocytosis, potocytosis, pinocytosis).

As used herein, the term "ligand" refers to a chemical compound or structure which will bind to a receptor. This includes but is not limited to ligands such as asialoorosomucoid, asialoglycoprotein, folate (compound A; FIG. 1), lipoic acid (compound G; FIG. 3), biotin (compound B; FIG. 2), apolipoprotein E sequence (Pep2; [SEQ ID NO:32] FIG. 18), compound D (FIG. 18), compound E (Asp (bis-LacAHT)) (FIG. 18), Fab' (FIG. 18), compound P L-tyrosyl-L-aspartoyl-bis-{N-[6-[[6-O-phosphoryl-α-D-mannopyranosyl]oxy]hexyl]-L-alaninamide] (FIG. 19), compound J 3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid, Pep12 (SEQ ID NO:42)-Pep19 (SEQ ID NO:49) (X is the 3(2-pyridyldithio)propionyl moiety (FIG. 20)), Pep12 (SEQ ID NO:42) ([Gln⁰, Leu²⁷, ε-X-Lys⁶⁷ ]-insulin-like growth factor II), Pep13 (SEQ ID NO:43) ([Gln⁰, ε-X-Lys³, Leu²⁷, Arg⁶⁷ ]-insulin-like growth factor II), Pep14 (SEQ ID NO:44) (Y⁰ -ε-X-Lys²⁴ -calcitonin gene-related peptide), Pep15 (SEQ ID NO:45) ([Asu²,7, Y⁸, ε-X-K²⁴ ]-calcitonin gene-related peptide), Pep16 (SEQ ID NO:46) ((Gln⁰, Leu²⁷, ε-X-Lys⁵⁴, Arg⁵⁵, Arg⁶⁷)-insulin-like growth factor II), Pep17 (SEQ ID NO:47) (N-X-des-(1-3)-[Arg⁶⁵, Arg⁶⁷ ]-insulin-like growth factor I), Pep18 (SEQ ID NO:48) (ε-X-K⁰ -thymopoietin), Pep19 (SEQ ID NO:49) (ε-X-K⁴ -thymopoietin, 7α,12α-dihydroxy-3β-(ω-aminoalkoxy)-5-β-cholan-24-oic acid), Pep20 (SEQ ID NO:50) (hepatocyte growth factor), Pep21 (SEQ ID NO:51) (endothelin-1), Pep22 (SEQ ID NO:52) (N-succinyl-[glu⁹,ala¹¹,15 ]-endothelin-1(8-21)), and Pep23 (SEQ ID NO:53) (r-atrial natriuretic factor). The ligand E can be used for delivering nucleic acid to hepatocytes and P for delivering nucleic acid to muscle cells.

One skilled in the art will readily recognize that the ligand chosen will depend on which receptor is being bound.

Since different types of cells have different receptors, this provides a method of targeting nucleic acid to specific cell types, depending on which cell surface ligand is used. Thus, the preferred cell surface ligand may depend on the targeted cell type.

The term "nuclear ligand" as used herein refers to a ligand which will bind a nuclear receptor. The term "nuclear receptor" as used herein refers to a chemical grouping on the nuclear membrane which will bind a specific ligand and help transport the ligand through the nuclear membrane. Nuclear receptors can be but are not limited to those receptors which bind nuclear localization sequences. Nonlimiting examples of nuclear ligands include those shown on FIG. 18 below, as well as, Pep3 (SEQ ID NO:33), Pep4 (SEQ ID NO:34), Pep5 (SEQ ID NO:35), Pep6 (SEQ ID NO:36), Pep7 (SEQ ID NO:37), Pep8 (SEQ ID NO:38), Pep9 (SEQ ID NO:39), and Pep10 (SEQ ID NO:40). In a preferred embodiment, the nuclear ligand, GYGPPKKKRKVEAPYKA(K)₄₀ WK (SEQ ID NO:60), is used to transport nucleic acid to the nucleus.

The term "lysis agent" as used herein refers to a molecule, compound, protein or peptide which is capable of breaking down an endosomal membrane and freeing the DNA transporter into the cytoplasm of the cell. This term includes but is not limited to viruses, synthetic compounds, lytic peptides, or derivatives thereof. The term "lytic peptide" refers to a chemical grouping which penetrates a membrane such that the structural organization and integrity of the membrane is lost. As a result of the presence of the lysis agent, the membrane undergoes lysis, fusion or both.

In the present invention, useful lysis agents include but are not limited to peptides of the Othromyxoviridae, Alphaviridae and Arenaviridae. Lysis agents also can include Pep24 (SEQ ID NO:54), Pep25 (SEQ ID NO:55), Pep26 (SEQ ID NO:56), any appropriate bacteria toxin, bacteria, adenovirus, parainflunza virus, herpes virus, retrovirus, hepatitis virus, or any appropriate lytic peptide or protein from a virus or bacteria. This includes use of any subfragments of the above which will provide endosomal escape activity. Particular bacterial toxins may include cytolytic toxins or active fragments from alveolysin, bifermentolysin, botulinolysin, capriciolysin, cereolysin O, chauveolysin, histolyticolysin O, ivanolysin, laterosporolysin, oedematolysin O, listeriolysin O, perfringolysin O, pneumolysin, sealigerolysin, septicolysin O, sordellilysin, streptoslysin O, tetanolysin or thuringolysin O.

The lysis agent can be a replication deficient virus. In one preferred embodiment, adenovirus of the structure F (FIG. 4) is used. As used herein, the term "replication deficient" refers to a virus lacking one or more of the necessary elements for replication. In another embodiment of the present invention, useful lytic peptides are Pep24 (SEQ ID NO:54)(influenza; GLFEAIAGFIEDGWEGMIDGGGC), Pep25 (SEQ ID NO:55)(SFV E1; KVYTGVYPFMWGGAYCFCD), and Pep26 (SEQ ID NO:56)(Lassa gp2; GGYCLTRWMLIEAELKCFGNTAV). In still another embodiment, bacteria toxins listeriolysin or perfringolysin can be used. The above are only examples and are nonlimiting.

Lysis agents as used herein are pH sensitive. After cointernalization of the nucleic acid complex containing the lysis agent throughout the same coated pit on the plasma membrane of the cell, the decrease in pH that occurs immediately after endosome formation causes spontaneous lysis of the endosome. The nucleic acid is then released into the cytoplasm. The above is a nonlimiting example.

The surface ligand, the nuclear ligand and/or the lysis agent can be attached directly to the binding molecule by covalent bonding or can be connected to the binding molecule via a spacer. The term "spacer" as used herein refers to a chemical structure which links two molecules to each other. The spacer normally binds each molecule on a different part of the spacer molecule. The spacer can be hydrophilic molecule and comprised of about 6 to 30 carbon atoms. The spacer can also contain between 6 to 16 carbon atoms. The spacer can include but is not limited to a hydrophilic, polymer of [(gly)_(i) (ser)_(j) ]_(k) wherein i ranges from 1 to 6, j ranges from 1 to 6, and k ranges from 3 to 20. In addition, the spacer and binding molecule compounds include but are not limited to those compounds expressed herein as XI, XII, XL, XLI, LX, LXI, LXXVIII, XC, CXVIV, CXVI, XV, XVI, XVIII, XXI, XLV, LXVII, XLVII, L, LXV, LXX, XCIV, XCVI, XCIX, XXIV, XXV, LXXIII, LXXIV, CII, or CV. Furthermore, the spacer may include but is not limited to repeating omega-amino acid of the structure [NH--(CH₂ CH₂)_(n) --CO--]_(m), where n=1-3 and m=1-20, a disulfide structure (CH₂ CH₂ --S--S--CH₂ CH₂ --)_(n), or an acid sensitive bifunctional molecule with the structure ##STR1##

A second aspect of the present invention is a nucleic acid transporter system for delivering nucleic acid into a cell. The transporter includes a nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and covalently linked to a surface ligand. In addition, the transporter includes a second nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and covalently linked to a nuclear ligand. The nucleic acid binding complexes can be non-covalently bound to the nucleic acid at the same time, i.e., simultaneously, and in various proportions. The binding molecules can be the same molecule or a combination of a different molecule as discussed above. Furthermore, the surface ligand and nuclear ligand can be directly attached to the binding molecule or attached by a spacer as defined above. In one embodiment of the present invention, the surface ligand can be one of those disclosed herein and the nuclear ligand is the peptide GYGPPKKRRKVEAPYKA(K)₄₀ WK (SEQ ID NO:60).

In a third aspect, the present invention features a nucleic acid transporter for delivering nucleic acid into a cell. The transporter includes a nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and covalently linked to a surface ligand. The transporter includes a second nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and covalently linked to a nuclear ligand. In addition, the transporter includes a third nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and covalently linked to a lysis agent. The binding complexes above can be noncovalently bound to the nucleic acid at the same time, i.e., simultaneously, and in various proportions. As described above, the binding molecules can be the same or different molecules and the binding molecules may attach to the ligands or lysis agent directly or by spacers.

A fourth related aspect of the present invention features a nucleic acid transporter system with a nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and covalently linked to a surface ligand, and a second nucleic acid binding complex with a binding molecule noncovalently bound to nucleic acid and covalently linked to a lysis agent. In one preferred embodiment, folate is used as the surface ligand and replication-deficient adenovirus is used as the lysis agent. This transporter, as well as the other nucleic acid transporters described in this invention, can deliver to the cytosol other macromolecules besides nucleic acid including but not limited to, proteins, lipids and carbohydrates. The binding complexes of this aspect can be noncovalently bound to the nucleic acid at the same time, i.e., simultaneously, and in various proportions. The binding molecules can be the same or different and may attach to the ligands or lysis gents directly or by spacers as described above.

In another preferred embodiment an asialoglycoprotein can be used as the surface agent and listeriolysin or perfringolysin as the lysis agent. Listeriolysin, perfringolysin or only a part of the toxins harboring the active subfragments need be used. Similarly, all microbial toxins and their active subfragments can be incorporated into the transporters of the present invention for endosomal escape.

In addition, a fifth related aspect features a nucleic acid transporter system containing a plurality of a common nucleic acid binding complex with a binding molecule noncovalently bound to nucleic acid and attached to both a surface ligand and a nuclear ligand. A lysis agent may also be bound to the binding molecule along with the surface ligand and nuclear ligand. As above, spacers can be used to connect the surface ligand, nuclear ligand or lysis agent.

A sixth aspect of the present invention features a cell transformed with the nucleic acid transporter system as described above for expression of nucleic acid targeted to the cell. As defined above, the nucleic acid may include nucleic acid containing genetic material and coding for a variety of proteins, polypeptides or RNA.

As used herein "transformation" is a mechanism of gene transfer which involves the uptake of nucleic acid by a cell or organism. Following entry into the cell, the transforming nucleic acid may recombine with that of the host or may replicate independently as a plasmid or a temperate phage. Cells which are able to take up nucleic acid are described as competent. Particular cells may not be naturally competent, but require various treatments in order to induce the transfer of nucleic acid across the cell membrane. This would include but is not limited to the surface ligand receptor interaction of the present invention.

Transformation can be performed by in vivo techniques as described below, or by ex vivo techniques in which cells are cotransfected with a nucleic acid transporter system containing nucleic acid and also containing a selectable marker. This selectable marker is used to select those cells which have become transformed. It is well known to those skilled in the art the type of selectable markers to be used with transformation studies.

The transformed cells can produce a variety of compounds selected from proteins, polypeptides or RNA, including hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, tumor antigens, viral antigens, parasytic antigens, and bacterial antigens. Other examples can be found above in the discussion of nucleic acid. The product expressed by the transformed cell depends on the nucleic acid used. The above are only examples and are not meant to be limiting.

A seventh aspect of the present invention features methods for transformation of cells. These methods comprise the steps of contacting a cell with a nucleic acid transporter system as described above for a sufficient time to transform the cell. Cell types of interest can include but are not limited to liver, muscle, endothelium and skin.

In an eighth aspect, the present invention features a transgenic animal whose cells contain the nucleic acid referenced above delivered via the nucleic acid transporter system. These cells include germ or somatic cells. Transgenic animal models can be used for dissection of molecular carcinogenesis and disease, assessing potential chemical and physical carcinogens and tumor promoters, exploring model therapeutic avenues and livestock agricultural purposes.

The genetic material which is incorporated into the cells from the above nucleic acid transporter system includes (1) nucleic acid not normally found in the cells; (2) nucleic acid which is normally found in the cells but not expressed at physiological significant levels; (3) nucleic acid normally found in the cells and normally expressed at physiological desired levels; (4) other nucleic acid which can be modified for expression in cells; and (5) any combination of the above.

A ninth related aspect of the present invention features compounds relating to the nucleic acid transporter systems above. These include but are not limited to compounds which are related to the nucleic acid binding complex, the binding molecule, surface ligands, nuclear ligands or lysis agents. These compounds are described below in more detail.

A tenth related aspect of the present invention features a method for delivering nucleic acid into a hepatocyte. This method includes contacting a hepatocyte with the above referenced nucleic acid transporters. The surface ligand used with the nucleic acid transporter is one specific for recognition by hepatocyte receptors. In particular, the asialoorosomucoid protein is used as a cell surface ligand, spermine derivative as a binding molecule and a replication-defective adenovirus as a lysis agent. The term "hepatocyte" as used herein refers to cells of the liver.

An eleventh related aspect of the present invention features a method for delivering nucleic acid to muscle cells. This method includes contacting the muscle cell with the above referenced nucleic acid transporter system. The surface ligand used is specific for receptors contained on the muscle cell. In particular, the surface ligand can be insulin-like growth factor-I. In addition, the binding molecule can be a spermine derivative and the lysis agent can be a replication-defective adenovirus. The term "muscle cell" as used herein refers to cells associated with striated muscle, smooth muscle or cardiac muscle.

A twelfth related aspect of the present invention features a method for delivering nucleic acid to bone-forming cells. This method includes contacting the bone-forming cell with the above-referenced nucleic acid transporter system. The surface ligand used with the nucleic acid transporter system is specific for receptors associated with bone-forming cells. In particular, the surface ligands can include but are not limited to bone morphogenetic protein or cartilage induction factor. In addition, the binding molecule of the nucleic acid transporter can be a spermidine derivative and the lysis agent a replication defective adenovirus. As used herein the term "bone-forming cell" refers to those cells which promote bone growth. Nonlimiting examples include osteoblasts, stromal cells, inducible osteoprogenitor cells, determined osteoprogenitor cells, chondrocytes, as well as other cells capable of aiding bone formation.

Another related aspect of the present invention features a method for delivering nucleic acid to a cell using the above-referenced nucleic acid transporter system. The nucleic acid transporter system uses folate as a ligand. In addition, the nucleic acid transporter can use a replication defective adenovirus as a lysis agent and as a binding molecule a spermine derivative. This method targets cells which contain folate receptors, including but not limited to, hepatocytes.

The nucleic acid transporters of the above methods may be administered by various routes. The term "administration" refers to the route of introduction of the nucleic acid transporter or carrier of the transporter into the body. Administration may be intravenous, intramuscular, topical, or oral. Administration can be directly to a target tissue or through systemic delivery. In particular, administration may be by direct injection to the cells. In another embodiment, administration may be intravenously. Routes of administration include intramuscular, aerosol, oral, topical, systemic, ocular, intraperitoneal and/or intrathecal.

Other features and advantages of the invention will be apparent from the following detailed description of the invention in conjunction with the accompanying drawings and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 show the schematic synthesis of the receptor ligands.

FIG. 4 shows a schematic synthesis of the virus ligand.

FIGS. 5-13 show a schematic flow chart for the synthesis of the nucleic acid transporter systems.

FIG. 14 is a schematic diagram of insertion of a triplex forming oligonucleotide or peptidyl nucleic acid by attachment of a ligand.

FIG. 15A is a schematic representation of using a ligand to target to a triplex forming oligonucleotide to a duplex nucleic acid. FIG. 15A is a schematic representation using a ligand to target a triplex forming peptidyl oligonucleotide to a duplex nucleic acid (Double stranded DNA sequences (l.) represent Seq. ID Nos. 1, 4, 7, and 10 respectively top to bottom of page; single stranded nucleic acid sequences (middle) represent Seq. ID Nos. 2, 5, 8, 11, and 14 respectively top to bottom of page; single stranded nucleic acid sequences (right) represent Seq. ID Nos. 3, 6, 7 or 9, 12, and 15 respectively top to bottom of page). FIG. 15B depicts a specific ligand for targeting muscle. FIG. 15C shows analogs useful in the compound of 15A. FIG. 15D depicts an analog of 15A.

FIG. 16 shows a specific targeting of a ligand for the SV40 sequences (single stranded nucleic acid sequence (top of page.) represents Seq. ID No.16; ten peptide sequence (middle of page) represents Seq. ID No.17; sixteen peptide sequence (bottom of page) represents Seq.ID No.18).

FIG. 17 shows the targeting with a ligand of a sequence to the c-myc promoter region (single stranded nucleic acid sequences (top of page.) represent Seq. ID Nos. 19, 20, and 21 respectively (reading top to bottom); peptide sequence (bottom of page) represents Seq. ID Nos. 22-29 respectively (reading top to bottom)).

FIG. 18 is a schematic of the peptide ligands and other ligands and shows the abbreviations used herein for the ligands.

FIG. 19 shows the schematic synthesis of receptor ligands.

FIGS. 20A and 20B shows specific ligands for targeting to muscle.

FIG. 21 shows the folyl-spermine derivative.

FIG. 22 shows a specific ligand for targeting muscle.

FIGS. 23A and 23B is a schematic of the peptide ligands and other ligands and shows the abbreviations used herein for the ligands.

FIG. 24 is a schematic representation of a synthetic route for bifunctional acid sensitive linkers.

FIGS. 25A, 25B, 25C, and 25D is a schematic representation of a trimeric fusogenic peptide.

FIGS. 26A and 26B is a schematic representation of the synthetic route for a trimeric fusogenic peptide.

FIG. 27 is a schematic representation of a synthetic route for a monomeric fusogenic peptide.

FIGS. 28A, 28B, and 28C is a schematic representation of a synthetic route for a trimeric fusogenic peptide.

FIGS. 29A, 29B, and 29C is a schematic representation of a synthetic route for a trimeric fusogenic peptide.

FIG. 30 is a time course analysis of ³ H-folic acid uptake in KB cells.

FIG. 31 is a time course analysis of ¹²⁵ I-BSA folate uptake in KB cells.

FIG. 32 is a quantitation of β-galactosidase expression in KB after gene delivery.

FIG. 33 is a quantitation of folate mediated gene delivery in tumor cells.

FIG. 34 is a representation of Factor IX expression after nucleic acid delivery to primary hepatocytes by ADV-PLL/DNA complexes.

FIG. 35 is a representation of β-galactosidase expression after nucleic acid delivery by Transferrin/DNA/PFO complexes.

FIG. 36 is a representation of liposome leakage using a monomeric form of the HA₂ -fusiogenic peptide.

FIG. 37 is a representation of liposome leakage using a dimeric form of the HA₂ -fusiogenic peptide.

The drawings are not necessarily to scale. Certain features of the invention may be exaggerated in scale or shown in schematic form in the interest of clarity and conciseness.

DETAILED DESCRIPTION OF THE INVENTION

The following are examples of the present invention using nucleic acid transporter systems for delivery of nucleic acid to a cell. These examples are offered by way of illustration and are not intended to limit the invention in any manner.

The following are specific examples of preferred embodiments of the present invention. These examples demonstrate how specific surface and nuclear ligands can be used with a nucleic acid binding moiety to target nucleic acid into the cellular interior and/or the cell nucleus. Furthermore, these examples demonstrate use of a lysis agent to release nucleic acid into the cellular interior. These examples include in vivo and in vitro techniques, various cellular or animal models and how nucleic acid can be inserted into cells. The utility of such nucleic acid transporter systems is noted herein and is amplified upon in copending applications by Woo et al., entitled "A DNA Transporter System and Method of Use", supra, and such sections are hereby specifically incorporated by reference herein.

Below are provided examples of specific nucleic acid transporter systems that can be used to provide certain functionalities to the associated nucleic acid in the nucleic acid transporter system, and thus within a transformed cell or animal containing such associated nucleic acid. Those in the art will recognize that specific moieties of the nucleic acid transporter system can be identified as that containing the functional region providing the desirable properties of the nucleic acid transporter system. Such regions can be readily minimized using routine deletion, mutation, or modification techniques or their equivalent.

Synthesis of Components of Receptor Ligands

Examples of the specific components of the receptor ligands are shown in FIGS. 1-4, 18.

A. In FIGS. 1-3 the schematic synthesis of A, B, G, P, J and M are shown. The actual synthesis for A, B, G, P, J and M are very similar. As an example, for the preparation of A, dissolve 1 mmol of folic acid in 2 ml dry dimethylformamide (DMF), add 1.3 mmol 1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir in the dark under N₂ overnight at 4°, then add 1.3 mmol N-hydroxysuccinimide with stirring continued for another 6 hr at 4°. Add bis(2-aminoethane)disulfide, 1 mmol in 0.5 ml dry dimethylformamide, dropwise to the reaction mixture and stir for an additional 4 hr. Add 15 ml water to precipitate the product. After centrifugation, the precipitate is washed and dissolved in oxygen-free 0.1 M NH₄ OH. This solution is applied to an anion exchange resin and equilibrated in degassed 0.05 M NH₄ CO₃ containing 20% acetonitrile. The γ-isomer is separated from unreacted starting materials and the α-isomer by chromatography in 0.1 M NH₄ CO₃ containing 20% acetonitrile. The appropriate fractions are pooled and lyophilized to obtain the product.

The synthesis of the nicotinic acetylcholine receptor ligand, component J, 3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid, is the same as for folic acid, component A, except the 1 mmol of folic acid in 2 ml dry DMF is replaced with 1 mmol 3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid in 2 ml dry DMF. The synthesis of the nicotinic acetylcholine receptor ligand, component J, 3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid, is the same as for folic acid, component A except the 1 mmol of folic acid in 2 ml dry DMF is replaced with 1 mmol 3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid in 2 ml dry DMF.

This compound can be further reacted to yield A'. Dissolve 2 mmol A in 10 ml oxygen-free 0.01 M NH₄ CO₃ containing 2 mmol dithioerythritol, stir for 2 hr. The solution is applied to an anion exchange resin equilibrated in degassed 0.1 M NH₄ CO₃ containing 20% acetonitrile. The reduced folate derivative is separated from unreacted starting materials by chromatography in 0.1 M NH₄ CO₃ containing 20% acetonitrile. The appropriate fractions are pooled, lyophilized, and then dissolved in 10 ml dry dimethylformamide for dropwise addition to a vigorously stirred solution of 2,2'-dipyridinedisulfide, 4 mmol dissolved in 10 ml ethanol containing 0.4 ml glacial acetic acid. After overnight at room temperature protected from light, the solvent is removed in vacuo. Add degassed 0.1 M NH₄ CO₃ to effect solution and then chromatograph as before to obtain the desired product. Both the original A, B and G and the further reacted A', B' and G' have been used. The synthesis of the nicotinic acetylcholine receptor ligand, component J, 3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid is the same as for folic acid, component A except the 1 mmol of folic acid in 2 ml dry DMF is replaced with 1 mmol 3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid in 2 ml dry DMF.

One skilled in the art will recognize that other vitamins and analogs of these vitamins can be used. Since the different vitamins and analogs will have different affinities, uptake and selectivity for the membrane receptors, the specific vitamin or analog is chosen to maximize the specificity and uptake.

B. The peptides including Pep1 (SEQ ID NO:31) through Pep11 (SEQ ID NO:41) and Pep21 (SEQ ID NO:51) through Pep26 (SEQ ID NO:56) can be synthesized by a variety of methods. In the present invention solid phase synthesis on a support is preferred, except for Pep12 (SEQ ID NO:42), Pep13 (SEQ ID NO:43), Pep16 (SEQ ID NO:46), Pep17 (SEQ ID NO:47) and Pep20 (SEQ ID NO:50) which are recombinant proteins produced by expression vectors in bacteria, yeast, baclovirus or mammalian systems.

Peptides Pep1 (SEQ ID NO:31) through Pep6 (SEQ ID NO:36) and Pep12 (SEQ ID NO:42) through Pep23 (SEQ ID NO:48) are examples of peptides or peptide analogs. These peptides target and bind to membrane receptors. One skilled in the art recognizes that other peptides or analogs to other membrane receptors can be used, and that the order of the amino acid sequence can be reversed, inverted and/or repeated, while still maintaining the transporter characteristics. The selection of a specific peptide will depend on the tissue and membrane receptor which is targeted. By selecting specific peptides, one skilled in the art recognizes the binding efficiency, uptake and specificity can be regulated. This can be used for tissue specificity.

Peptides Pep24 (SEQ ID NO:54) through Pep26 (SEQ ID NO:56) are examples of peptides or peptide analogs. These peptides lyse membranes. One skilled in the art recognizes that other peptides or analogs to other lytic peptides can be used, and that the order of the amino acid sequence can be reversed, inverted and/or repeated, and still maintain the lytic characteristics. The selection of a specific peptide will depend on the tissue and membrane receptor which is be targeted.

C. Spermine Template

C1. Monomeric Fusogenic Peptide Covalently Linked to a Polycation Through an Acid Sensitive, Reducible Spacer ##STR2## where R=Pep24 (SEQ ID NO:54) --SS--CH₂ CH₂ NH--α--CO-aconityl-γ--CO-- ##STR3## where R=GLFEAIADFIENGWEGMIDGGGC--SS--CH₂ CH₂ NH--α--CO-aconityl-γ--CO--(SEQ ID NO:54)

Detailed preparative procedures are as follows: Combine 2 mmol (S)-hydroxyspermine (IV), 4 mmol 4-pyrrolidinopyridine and 4.1 mmol 9-fluorenylmethyl chloroformate in 40 ml anhydrous benzene and stir overnight at room temperature under N₂. Separate the desired product (1) by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product as an amorphous solid.

Add 4 ml of dry benzene to 1 mmol K₂ CO₃ and 2 mmol 18-crown-6 and stir for 20 min. Add 2 mmol of 1 in 4 ml of dry benzene, followed by 2 mmol methyl bromoacetate in 2 ml benzene After 4 hr, add 25 ml of water and extract with 3 portions of 25 ml benzene. Remove the solvent in vacuo and dissolve the residue in 10 ml ethanol containing 2 mmol potassium hydroxide. After overnight at room temperature, the solution is transferred to a separatory funnel, to which 2 mmol of HCl, 5 ml of water and 25 ml of benzene is added.

After extraction with 3 additional portions of benzene, the combined organic phase is taken to dryness in vacuo, redissolved in a minimum volume of ethyl acetate, diluted with enough petroleum ether to create slight turbidity and cooled at 4° to promote crystallization of 3.

Dissolve 1 mmol of 3 in 2 ml dry dimethylformamide, add 3.0 mmol 1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hr, then add 1.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr at room temperature. This solution is added dropwise to 10 mmol of 1,2-diaminoethane in 0.5 ml dry dimethylformamide, and stirring continued for an additional 4 hr, when the reaction is complete as monitored by thin layer chromatography. The solution is applied to an cation exchange resin equilibrated in degassed water. The product is separated from unreacted starting materials by a gradient from 0.0005 to 2.0 M HCl. The appropriate fractions are pooled and lyophilized to obtain the product (4).

The Synthetic route for the bifunctional acid sensitive linker 6 is shown in FIG. 24. Detailed procedures are as follows:

Combine 10 mmol cis-aconityl anhydride 10 mmol with 2-(2'-aminoethyldithio)-pyridine in 20 ml dry DMF under N₂ protected from light at room temperature for 18 hr. Remove the solvent in vacuo, dissolve the residue in the minimum amout of 3M NH₄ CO₃, dilute 10 fold and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ and elute with a gradient to 1.0M NH₄ CO₃. The appropriate fractions are pooled, lyophilized and the residue 5 dissolved in 2-propanol and crystallized at 4° after the solution is made turbid by the addition of diethyl ether. The product 5 is collected by filtration.

Dissolve 1 mmol of 5 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimide and 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another 24 hr at 4°. Remove the solvent in vacuo, dissolve the residue in the minimum amount of 2-propanol and crystallize at 4° after the solution is made turbid by the addition of diethyl ether. The product 6 is collected by filtration.

Dissolve 1 mmol of 6 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimide and 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another 6 hr at 4°. One mmol of 4 in 0.5 ml dry dimethylformamide, is added dropwise to the preceding reaction mixture and stirring continued for an additional 4 hr. Remove the solvent in vacuo and dissolve the residue in 10 ml 50% piperidine in DMF (v/v). Again remove the solvents in vacuo, solubilize the residue 7 in 0.1 M NH₄ OH, and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ containing 20% acetonitrile and eluted with a gradient of 0 to 0.25 M acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 8.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 (SEQ ID NO. 54) in 5 ml PBS, pH 7.4, at 4°, add 0.1 mmol of 8 in PBS dropwise. After 18 hrs, chromatograph over a molecular sieve to separate the product 9 from unreacted starting materials.

C2. Trimeric Fusogenic Peptide Covalently Linked to a Polycation Through an Acid Sensitive, Reducible Spacer ##STR4## where R=--α--CO--aconityl--γ--COHNC--(OCH₂ CH₂ NHCO(CH₂)₅ NH--R')₃ and R'=Pep24 (SEQ ID NO:54)--SS--CH₂ CH₂ CO--

The Series II synthetic route (New compounds 10-16) is shown in FIG. 25. Detailed procedures are as follows.

Add 4 ml of dry benzene to 1 mmol K₂ CO₃ and 2 mmol 18-crown-6 and stir for 20 min. Add 2 mmol N-t-BOC-tris-(hydroxymethyl)methane in 4 ml of dry benzene, followed by 20 mmol 1-bromo-2-(N-5-FMOC-aminohexanoyl)-aminoethane in 2 ml benzene. After 4 hr, add 25 ml of water and extract with 3 portions of 25 ml benzene. Remove the solvent in vacuo and dissolve the residue 10 in 10 ml 50% piperidine in DMF (v/v). After 6 hr, remove the solvents in vacuo, solubilize the residue in 20 ml ethyl acetate, wash with water until neutral and dry over molecular sieve before solid phase extraction and chromatography on octadecyl-silica, using a gradient of acetonitrile to 100%. Pool the appropriate fractions to obtain 11.

Combine 2 mmol of 11 in 20 ml acetonitrile with 7 mmol of succinimidyl 3(2-pyridyldithio)-propionate in ethanol. After 60 min, dilute with sufficient water to create a slight turbidity and apply to octadecyl-silica, again using a gradient of acetonitrile from 0 to 100%. Pool the appropriate fractions to obtain 12.

Dissolve 1 mmol of 12 in 20 ml 3N HCl at 4° and allow to stand for 60 min before the solution is taken to dryness in vacuo. The residue is resuspended in water and solubilized with the minimum amount of acetonitrile and chromatographed on octadecyl-silica, again using a gradient of acetonitrile from 0 to 100%. Pool the appropriate fractions to obtain 13.

Dissolve 1 mmol of 13 in 2 ml dry DMF, add 3 mmol cis-aconityl anhydride and stir under N₂ overnight at 4°. Remove the solvents in vacuo, solubilize the residue in 0.1 M NH₄ OH, and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ containing 20% acetonitrile and eluted with a gradient of 0 to 0.25 M acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 14.

Dissolve 1 mmol of 14 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimide and 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another 6 hr at 4°. One mmol of 4 in 0.5 ml dry dimethylformamide, is added dropwise to the preceding reaction mixture and stirring continued for an additional 4 hr. Remove the solvent in vacuo and dissolve the residue in 10 ml 50% piperidine in DMF (v/v). Again remove the solvents in vacuo, solubilize the residue in 0.1 M NH₄ OH, and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ containing 20% acetonitrile and eluted with a gradient of 0 to 0.25 M acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 15.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 (SEQ ID NO:54) in 5 ml PBS, pH 7.4, at 4°, add 0.1 mmol of 15 in PBS dropwise. After 18 hr, chromatograph over a molecular sieve to separate the product 16 from unreacted starting materials.

C3. Trimeric Fusogenic Peptide Covalently Linked to a Polycation Through an Acid Sensitive, Reducible Spacer ##STR5## where R"= ##STR6## with the γ-carboxyl of glu¹ in amide linkage with the γ-amino of α,γ-diaminobutyric acid¹⁰ and R'=Pep24--SS--CH₂ CH₂ CO--

The Series III synthetic route (New compounds 17-20) is shown in FIG. 26. Detailed experimental procedures are as follows.

Solid phase peptide synthesis with conventional reagents and procedures gives 17. It is obvious to one skilled in the art that homologs of 2,4-diaminobutyric acid, such as orinithine and lysine, could be substituted for this residue and that other amino acids, such as serine, alanine, and aspartic acid, could be substituted for gly.

Dissolve 1 mmol of 17 in 2 ml dry DMF, add 3 mmol cis-aconityl anhydride and stir under N₂ overnight at 4°. Remove the solvents in vacuo, solubilize the residue in 0.1 M NH₄ OH, and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ containing 20% acetonitrile and eluted with a gradient of 0 to 0.25 M acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 18.

Dissolve 1 mmol of 18 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimide and 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another 6 hr at 4°. One mmol of 4 in 0.5 ml dry dimethylformamide, is added dropwise to the preceding reaction mixture and stirring continued for an additional 4 hr. Remove the solvent in vacuo and dissolve the residue in 10 ml 50% piperidine in DMF (v/v). Again remove the solvents in vacuo, solubilize the residue in 0.1 M NH₄ OH, and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ containing 20% acetonitrile and eluted with a gradient of 0 to 0.25 M acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 19.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 (SEQ ID NO:54) in 5 ml PBS, pH 7.4, at 4°, add 0.1 mmol of 19 in PBS dropwise. After 18 hr, chromatograph over a molecular sieve to separate the product 20 from unreacted starting materials.

C4. Monomeric Fusogenic Peptide Covalently Linked to a Polycation Through an Acid Sensitive, Reducible Spacer ##STR7## where R=Pep24 (SEQ ID NO:54) --SS--CH₂ CH₂ NH--α--CO--aconityl--γ--CO--

The Series IV synthetic route (New compounds 21-24) is shown in FIG. 27. Detailed experimental procedures are as follows.

Solid phase peptide synthesis with conventional reagents and procedures gives 21. Dissolve 1 mmol of 21 in 2 ml dry DMF, add 3 ml of 6 and stir under N₂ overnight at 4°. Remove the solvents in vacuo, solubilize the residue in acetonitrile and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ containing 20% acetonitrile and eluted with a gradient of 0 to 1.0 M acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 22.

Dissolve 1 mmol of 22 in 10 ml 50% piperidine in DMF (v/v). Remove the solvents in vacuo, solubilize the residue in 0.1 M NH₄ OH, and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ and elute with a gradient to 1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 23.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 (SEQ ID NO:54) in 5 ml PBS, pH 7.4, at 4°, add 0.1 mmol of 23 in PBS dropwise. After 18 hrs, chromatograph over a molecular sizing column to separate the product 24 from unreacted starting materials.

C5. Trimeric Fusogenic Peptide Covalently Linked to a Polycation Through an Acid Sensitive, Reducible Spacer ##STR8## where R=--α--CO--aconityl--γ--COHNC--(OCH₂ CH₂ NHCO(CH₂)₅ NH--R')₃ and R'=Pep24 (SEQ ID NO:54) --SS--CH₂ CH₂ CO--

The Series V synthetic route (New compounds 25-27) is shown in FIG. 28. Detailed experimental procedures are as follows.

Dissolve 1 mmol of 14 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimide and 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another 6 hr at 4°. One mmol of 21 in 0.5 ml dry dimethylformamide, is added dropwise to the preceding reaction mixture and stirring continued under N₂ overnight at 4°. Remove the solvents in vacuo, solubilize the residue in acetonitrile and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ containing 20% acetonitrile and eluted with a gradient of 0 to 1.0 M acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 25.

Dissolve 1 mmol of 25 in 10 ml 50% piperidine in DMF (v/v). Remove the solvents in vacuo, solubilize the residue in 0.1 M NH₄ OH, and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ and elute with a gradient to 1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 26.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 (SEQ ID NO:54) in 5 ml PBS, pH 7.4, at 4°, add 0.1 mmol of 23 in PBS dropwise. After 18 hr, chromatograph over a molecular sizing column to separate the product 27 from unreacted starting materials.

C6. Trimeric Fusogenic Peptide Covalently Linked to a Polycation Through an Acid Sensitive, Reducible Spacer ##STR9## where R"= ##STR10## with the γ-carboxyl of glu¹ in amide linkage with the γ-amino of α,γ-diaminobutyric acid¹⁰ and R'=Pep24 (SEQ ID NO:54) --SS--CH₂ CH₂ CO--

The Series VI synthetic route (New compounds 28-30) is shown in FIG. 29. Detailed experimental procedures are as follows.

Dissolve 1 mmol of 18 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimide and 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another 6 hr at 4°. One mmol of 21 in 0.5 ml dry dimethylformamide, is added dropwise to the preceding reaction mixture and stirring continued under N₂ overnight at 4°. Remove the solvents in vacuo, solubilize the residue in acetonitrile and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ containing 20% acetonitrile and eluted with a gradient of 0 to 1.0 M acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 28.

Dissolve 1 mmol of 28 in 10 ml 50% piperidine in DMF (v/v). Remove the solvents in vacuo, solubilize the residue in 0.1 M NH₄ OH, and apply to an anion exchange resin equilibrated in degassed 0.05 M NH₄ CO₃ and elute with a gradient to 1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product 29.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 (SEQ ID NO:54) in 5 ml PES, pH 7.4, at 4°, add 0.1 mmol of 29 in PBS dropwise. After 18 hr, chromatograph over a molecular sizing column to separate the product 30 from unreacted starting materials.

D. Peptides such as Pep7 (SEQ ID NO:37) through Pep10 (SEQ ID NO:40) are nuclear localization sequences which are used to target the inserted nucleic acid to the nucleus. One skilled in the art recognizes that the peptides shown in FIG. 18 are only examples of this class of peptides and that there are a wide variety of other nuclear localization sequence peptides which can be used.

H₂ N-Tyr-ε-N-lys-Pep11 (SEQ ID NO:61) --CONH, and γ-N-[N-2-methoxy-6-chloroacridinyl-HN-tyr-ε-N-lys-Pep11 (SEQ ID NO:61) --CO]-2-N-(2-methoxy-6-chloroacridinyl) diaminobutanoyl-CONH₂ were prepared by standard solid phase peptide synthesis. One skilled in the art recognizes that any amino acid polymer such as H₂ N--(lys)_(n) (SEQ ID NO:62) --COOH, H₂ N--(arg-ala)_(n) (SEQ ID NO:63)--COOH, histones and other DNA binding cationic polypeptides and proteins which form an α-helix, can be substituted for the lys-ala template. The --NH--(lys-ala)_(n) (SEQ ID NO:63) --CO unit can be extended. The useful range is from 2 to greater than 100 depending on the sequence of the inserted DNA, the target, uptake and specificity. The sequence position of the ε-N-substituted-lys residue can be either amino-terminal or carboxyl-terminal. The substitution can be any amino reactive DNA binding dye as well as the acridine moiety. Examples of DNA binding dyes include thiaxanthenones, lucanthone, hycanthone, phenanthrenemethanol, metallointercalation reagents, tilorone, napthiophene, phenanthridiniums, dimidium, ethidium, propidium or quinacrine.

In further embodiments, the spacers can be attached to the α-amino group of the N-terminal amino acid, and/or the carboxyl group of the C-terminal amino acid, rather than the ε-amino group of lysine, to reduce immunological response to the ligand.

E. In addition to the above components, it has also been found that fusion competent virus can be used to target the inserted nucleic acid. FIG. 4 shows the schematic procedure for preparing a fusion competent virus for use in the present invention. A variety of fusion competent virus can be used. As an example, adenovirus can be prepared in two separate ways. To a stirred solution of 10 mg of fusion competent adenovirus in 5 ml PBS, pH 7.4, at 4°, add 0.3 ml of 20 mM succinimidyl 3(2-pyridyldithio)propionate in ethanol dropwise. After 60 min, dialyze against 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hr. Alternatively, to a stirred solution of 10 mg of fusion competent adenovirus in 5 ml PBS, pH 7.4, at 4°, add 0.3 ml of 20 mM 2-iminothiolane HCl in ethanol dropwise. After 60 min, dialyze against 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hr.

F. Recombinant Peptides 12 (SEQ ID NO:42), 13 (SEQ ID NO:43), and 16 (SEQ ID NO:46), 10 mg, prepared and purified by methods known in the art are dissolved in 10 ml 50 mM NH₄ OH, pH 8.5. Aliquots are removed at 2 hr intervals to determine the extent of cyclization of N-terminal glutamine to pyroglutamate. When this reaction is complete, the solution is lypholyzed and then dissolved in 5 ml PBS, pH 7.4. Peptides 12 (SEQ ID NO:42), 13 (SEQ ID NO:43), 16 (SEQ ID NO:46), and 17 (SEQ ID NO:47) are further reacted with either succinimyl 3(2-pyridyldithio) propionate or 2-iminothiolane as described for adenovirus above.

G. Further, it has been found that either monoclonal or polyclonal IgG can be used to target the inserted nucleic acid. Generally, the IgG is cleaved with immobilized pepsin to yield (Fab'--S--)₂ which is selectively reduced to Fab'--SH. Specifically, this includes: adding dropwise 0.5 ml 0.1 mM dithiothreitol to a stirred 5 ml solution of 1 nmol IgG F(ab')₂ at 4°, which was prepared by standard methods with immobilized pepsin. After 60 min, dialyze against 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hr.

H. Synthesis of component D. Standard continuous-flow solid phase synthetic methodologies are used to prepare H₂ N-his-leu-arg-arg-leu-arg-arg-arg-leu-leu-arg-glu-ala-glu-glu-gly--CONH.sub.2 (SEQ ID NO:18), which is released as a protected peptide, containing N^(im) -Fmoc-his, N⁹ -4-methoxy-2,3,6-trimethylphenylsulfonyl-arg, and glu-γ-Fmoc ester. Coupling of this protected peptide using DCCI with the appropriate protected peptide on the solid support, gives ##STR11## Deprotection of the asp-amino group, reaction with succinimidyl 3(2-pyridyldithio)propionate, deblocking and cleavage from the resin gives D.

I. Synthesis of the asialoglycoprotein receptor ligand, component E. Dissolve 1 mmol N.sup.α,N.sup.β -bis{hexanamido[tris-(β-lactosylhydroxymethyl)methane]}aspartyl diamide, in 20 ml PBS, pH 7.4, and combine with 1 mmol succinimyl 3(2-pyridyldithio) propionate, in 2 ml phosphate-buffered saline, pH 7.4. Dilute the reaction mixture 20-fold with water, apply to a cation exchange column to separate the desired product from unreacted starting material and other products, using a linear gradient formed from equal volumes of water and 2.0 M HCl. The appropriate fractions are pooled and lyophilized to obtain the product E.

J. Preparation of Compound I. L-tyrosyl-L-aspartoyl-bis-{N-[6-[[6-O-phosphoryl-α-D-mannopyranosyl]oxy]hexyl]-L-alaninamide] is prepared as described in the art, except that N-t-BOC-L-tyrosine is used in lieu of N-acetyl-L-tyrosine. Compound I is further reacted as described for compound E discussed above.

Synthesis of Tetracationic Nucleic Acid Binding Templates

The overall schematic flow chart for the synthesis of these compounds is shown in FIG. 5. The chemical pathway of synthesis is shown below. The Roman numerals are used to identify the specific compounds.

Dissolve 2 mmol of free base 1,4-diaminobutan-2-ol (II) in 5 ml of ethanol, add 4.1 mmol of acrylonitrile and allow to stand overnight at room temperature. Cool in an ice bath and saturate the solution with anhydrous NH₃ at 0°. Add about 5 ml of sponge nickel hydrogenation catalyst and shake under H₂ on a Paar low-pressure hydrogenator until the theoretical amount of H₂ is consumed. Remove the catalyst by filtration and wash the catalyst with ethanol. Combine filtrate and washings, then remove the ethanol in vacuo. Chromatograph on a cation exchange column to separate the desired products R, S and IV from unreacted starting material and other products, using a linear gradient formed from equal volumes of water and 2.0 M HCl. Resolve the enantiomers of IV on a chiral column such as (R)-N-3,5-dinitrobenzoylleucine-silica (Baker) by a gradient of 2-propanol, from 0 to 20% in hexane. ##STR12##

Next combine 2 mmol (S)-hydroxyspermine (IV), 8 mmol 4-pyrrolidinopyridine and 8.2 mmol benzyloxycarbonyl anhydride (Z₂ O) in 40 ml anhydrous benzene and stir overnight at room temperature under N₂. Separate the desired product V by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate from 0 to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product as an amorphous solid. ##STR13##

Add 4 ml of dry benzene to 1 mmol K₂ CO₃ and 2 mmol 18-crown-6 and stir for 20 min. Add 2 mmol V in 4 ml of dry benzene, followed by 2 mmol methyl bromoacetate in 2 ml benzene. After 4 hr, add 25 ml of water and extract with 3 portions of 25 ml benzene. Remove the solvent in vacuo and dissolve the residue IV in 10 ml ethanol containing 2 mmol potassium hydroxide. After overnight at room temperature, the solution is transferred to a separatory funnel, to which 2 mmol of HCl, 5 ml of water and 25 ml of benzene are added. After extraction with 3 additional portions of benzene, the combined organic phase is taken to dryness in vacuo, redissolved in a minimum volume of ethyl acetate, dried over 10% w/v anhydrous Na₂ SO₄ overnight. The organic phase is decanted and diluted with a sufficient amount of petroleum ether to create slight turbidity and cooled at 4° to promote crystallization of VII (1,4,9,12-tetrabenzyloxycarbonyl-1,12-diamino-6-carboxymethoxy-4,9-diazadodecane). ##STR14##

Dissolve 1 mmol of VII in 2 ml dry dimethylformamide, add 3.0 mmol 1-ethyl-3-[3-(dimethylamino)propyl)]carbodiimide, stir 2 hr, then add 1.1 mmol N-hydroxysuccinimide and stir for an additional 6 hr at room temperature. This solution is added dropwise to 1 mmol of A in 0.5 ml dry dimethylformamide. Stirring is continued in the dark under N₂ for an additional 4 hr. When the reaction is complete, as monitored by thin layer chromatography, 15 ml oxygen-free water is added to precipitate the product. The product is collected by centrifugation, washed and dissolved in oxygen-free 0.1 M NH₄ OH. The solution is applied to an anion exchange resin equilibrated in degassed 0.1 M NH₄ CO₃ containing 20% acetonitrile. The γ-isomer is separated from unreacted starting materials and the α-isomer by a gradient from 20% to 50% acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product. Alternative compounds in this series, for example VIIIb and VIIIc, are made by substituting the appropriate starting material containing biotin, lipoic acid or other substituent. ##STR15## Where R is A (VIIIa), B (VIIIb), G (VIIc) or H (IX).

Dissolve 1 mmol VIIIa or VIIIb or VIIIc or IX in 20 ml glacial acetic acid containing 30% HBr and stir overnight at room temperature in the dark under N₂. Add 30 ml diethyl ether to precipitate the product. Wash the product until the odor of acetic acid is gone. Dissolve the solid in oxygen-free 0.1 M NH₄ OH and apply to an anion exchange resin equilibrated in degassed 0.1 M NH₄ CO₃ containing 20% acetonitrile. The product Xa or Xb or Xc or XI is separated from unreacted starting materials by a gradient of 0 to 90% acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product. ##STR16## Where R is A (Xa), B (Xb , G (Xc) or H (XI).

Dissolve 2 mmol XI purified by chromatography as was done for IV, in 10 ml oxygen-free 0.01 M NH₄ CO₃ containing 2 mmol dithiothreitol and stir for 2 hr. Bring the solution to pH 5 with 1N HCl and apply the solution to a cation exchange resin equilibrated in degassed water. The product XII is isolated by a gradient from 0.0005 M to 2.0 M HCl. The appropriate fractions are pooled and lyophilized to obtain the product. This is followed by reactions described above to yield XIIId, XIIIe and XIIIf. ##STR17## Where R is D (XIIId), E (XIIIe) or F (XIIIf).

Dissolve 1 mmol XII in 2 ml phosphate-buffered saline, pH 7.4, and combine with 1 mmol of further reacted A, B or G (as described above) in 2 ml phosphate-buffered saline, pH 7.4. Dilute the reaction mixture 20-fold with water, apply to an anion exchange resin equilibrated in degassed 0.1 M NH₄ CO₃ containing 20% acetonitrile. The product XIa, XIb or XIc is separated from unreacted starting materials by chromatography in 0.1 M NH₄ CO₃ containing 20% acetonitrile. The appropriate fractions are pooled and lyophilized to obtain the product. ##STR18## Where R is A (Xia), B (Xib) or G (Xic).

Dissolve 1 mmol of VII in 2 ml dry dimethylformamide, add 3 mmol 1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hr, then add 1.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr at room temperature. This solution is added dropwise to 5 mmol of 1,6-diaminohexane in 20 ml dry dimethylformamide, and stirring continued for an additional 24 hr. Remove the solvent in vacuo. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate from 0 to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product as an amorphous solid (XIV). Next combine 1 mmol XIV in dry 10 ml benzene with 1.1 mmol succinimidyl 3(2-pyridylthio)propionate, stir for 2 hr at room temperature, and then remove the solvent I in vacuo. The resultant product is XV. ##STR19##

Dissolve 1 mmol XV, in 20 ml glacial acetic acid containing 30% Hbr and stir overnight at room temperature in the dark under N₂. Add 30 ml diethyl ether to precipitate the product. Wash the product until the odor of acetic acid is gone. Dissolve the solid in oxygen-free 0.1 M NH₄ OH. The solution is applied to an anion exchange resin equilibrated in degassed 0.1 M NH₄ CO₃ containing 20% acetonitrile. The product is separated from unreacted starting materials by a gradient of 20 to 80% acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product: ##STR20##

To a stirred solution of 10 mg of Fab'-SH in 5 ml PBS, pH 7.4, at 4°, add 0.3 ml of 10 mM XVI in ethanol dropwise. After 60 min. dialyze against 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hrs. ##STR21##

Combine 2 mmol IX, 2 mmol 4-pyrrolidinopyridine and 3 mmol succinic anhydride in 40 ml anhydrous benzene and stir overnight at room temperature under N₂. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product as an amorphous solid. This is followed with DCCI, in situ coupling with resin bound protected peptide, Pep2 (SEQ ID NO:32) --COOH, using standard solid phase synthetic techniques followed by deprotection and release from the resin to yield XIXh. ##STR22##

Dissolve 1 mmol of VII in 2 ml dry dimethylformamide, add 3.0 mmol 1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hr, then add 1.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr at room temperature. This solution is added dropwise to 5 mmol of methyl 6-amonohexanoate in 20 ml dry dimethylformamide, and stirring continued for an additional 24 hr. Remove the solvent in vacuo. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate from 0 to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product XX as an amorphous solid. ##STR23##

Dissolve 2 mmol XX in 10 ml ethanol containing 2 mmol potassium hydroxide. After overnight at room temperature, the solution is transferred to a separate funnel, to which 2 mmol of HCl, 5 ml of water and 25 ml of benzene is added. After extraction with 3 additional portions of benzene, the combined organic phase is taken to dryness in vacuo. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate from 0 to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product XXI as an amorphous solid. Next, couple XXI to the amino terminal of the peptide on the support using standard solid phase peptide methods. ##STR24## 1,4,9,12-tetrabenzyloxycarbonyl-1,12-diamino-6-[N(5'-carboxypentyl)aminocarbonylmethoxy]-4,9-diazadodecane ##STR25## Where R is: Pep3 (SEQ ID NO:33) (XXIIi), Pep4 (SEQ ID NO:34) (XXIIj), Pep5 (SEQ ID NO:35) (XXIIk) or Pep6 (SEQ ID NO:36) (XXIIl)

Further Tetracationic DNA Binding Templates

The overall schematic flow chart of the synthesis of these compounds is shown in FIG. 6. The chemical pathway of synthesis is shown below.

A. After derivatization of the ε-N-lys with succinic anhydride it is coupled to the ligands shown below:

    H.sub.2 N--CH.sub.2 CH.sub.2 --S--S--CH.sub.2 CH.sub.2 --NH--R

Where R is A, B, G or H.

Deprotection and release from the resin yields: ##STR26## Where R is A (XXIIIa), B (XXIIIb), G (XXIIIC) or H (XXIV).

Following the procedures described for the synthesis of XII and XIII but substituting XXIV for XI yields XXV and XXVI. ##STR27## Where R is D (XXVId), E (XXVIe) or F (XXVIf).

After derivatization of the ε-N-succinyl-lys with H₂ N--CH₂ CH₂ --S--S--CH₂ CH₂ --NH--t-BOC and deblocking, the ligand Pep2 (SEQ ID NO:32) is synthesized on the resin using standard solid phase techniques. Deblocking and cleavage from the resin yields XXVIIh. ##STR28##

The resin bound lys-ε-NH--CO(CH₂)₅ NH₂ intermediate was coupled with succinimidyl 3(2-pyridyldithio)propionate and then deprotected and cleaved to yield XXVIII. ##STR29##

To a stirred solution of 10 mg of FAB'--SH in 5 ml PBS, pH 7.4, at 4°, add 0.3 ml of 10 mM XVI in PBS, pH 7.4, dropwise. After 60 min. dialyze against 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hr. ##STR30##

The nuclear localization sequence was added by standard solid phase synthetic methods to the lys-ε-NH--CO(CH₂)₅ NH₂ intermediate of the resin bound protected peptide for carboxyl to amino orientation or to the N-succinyl derivative of ε-N-lys for amino to carboxyl orientation. Deprotection and release from the resin yields: ##STR31## Where R is Pep7 (SEQ ID NO:37) (XXXi), Pep8 (SEQ ID NO:38) (XXXj), Pep9 (SEQ ID NO:39) (XXXk) or Pep10 (SEQ ID NO:61) (SEQ ID NO:40) (XXXl) or ##STR32## Where R is Pep3 (SEQ ID NO:33) (XXXIi), Pep4 (SEQ ID NO:34) (XXXIj), Pep5 (SEQ ID NO:35) (XXXIk) or Pep6 (SEQ ID NO:36) (XXXIl)

B. Parent compound, N-(ligand moiety)-HN-tyr-lys-lys-ala-lys-ala-lys-ala-lys-CONH₂ (SEQ ID NO:61), was prepared by standard solid phase peptide synthesis. An amino acid polymer, such as H₂ N-(lys)_(n) (SEQ ID NO:62) --COOH, H₂ N-(arg-ala)_(n) (SEQ ID NO:63) --COOH, histones, and other nucleic acid binding cationic polypeptides and proteins which form an α-helix, can be used for the lys-ala template. The --HN-(lys-ala)_(n) (SEQ ID NO:64) --CO unit can be extended from 4 to more than 100. The sequence position of the residue bearing the spacer-ligand moiety can be either amino-terminal or carboxyl-terminal. In another embodiment, the ligand-spacer moiety is linked through a disulfide bond to cys as either the N-terminal or C-terminal residue.

The resin bound protected peptide containing a deblocked α-amino-tyr moiety is the synthetic intermediate for preparation of templates for reductive release of a plasma membrane receptor ligand. After derivatization of the α-amino-tyr with succinic anhydride, the following ligands are coupled to the resin bound protected peptide.

    H.sub.2 N--CH.sub.2 CH.sub.2 --S--S--CH.sub.2 CH.sub.2 --NH--R

where

    R=γ-amide of the glutamyl moiety of folic acid       (A)

    =biotin                                                    (B)

    =lipoic acid                                               (G)

    =H

Deprotection and release from the resin gives: ##STR33## where

    R=γ-amide of the glutamyl moiety of folic acid       XXIIIa

    =biotin                                                    XXIIIb

    =lipoic acid                                               XXIIIc

    =H

Synthesis of Hexacationic DNA Binding Template

A schematic flow chart of the synthesis of these compounds is shown in FIG. 7. The chemical pathway of synthesis is shown below.

Dissolve 2 mmol of succinic monoamide in 2 ml dry DMF, add 4.0 mmol 1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hr. Then add 2.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr at room temperature.

Combine this in dropwise fashion to 1 mmol (S)-hydroxyspermine (IV) in 2 ml dry DMF. After stirring overnight at room temperature, remove the solvent in vacuo, dissolve in water, and apply the solution to a cation exchange resin. The product is isolated by a gradient to 2.0 HCl. The appropriate fractions are pooled and lyophilized to obtain XXXII.

Dissolve 2 mmol XXXII in 5 ml dry toluene and add 10 mmol sodium bis(2-methoxyethoxy)aluminum hydride in toluene in small aliquots over 30 min. After 2 hr, add 10 ml ethyl acetate, then remove the solvents in vacuo. Dissolve the solids in water, adjust the pH to 3 with HCl and apply to a cation exchange resin. The product is isolated by a gradient to 2.0 M HCl. The appropriate fractions are pooled and lyophilized to obtain the product. ##STR34##

It is obvious to one skilled in the art that homologs with additional --(CH₂)₄ --NH₂, or --(CH₂)₃ --NH₂ units can be made by repeating the reactions using XXXIII in lieu of IV with H₂ NCOCH₂ CH₂ COOH or CH₂ ═CHCN.

In reactions similar to the above reaction where IV is converted to VII, XXXIII can be converted to XXVI. ##STR35## 1,5,9,14,18,22-hexabenzyloxycarbonyl-1,22-diamino-11-carboxymethoxy-5,9,14,18-tetraazadocosane

In reactions similar to the above reactions where VII is converted to the VII and XI series, XXXVI can be converted to the XXXVII and XXXIX series. ##STR36## Where R is A (XXXVIIa), B (XXXVIIb), G (XXXVIIc) or H (XXXVIII). ##STR37## Where R is A (XXXIXa), B (XXXIXb), G (XXXIXc) or H (XL).

In reactions similar to the above for the conversion of XI to the XIII series XL is converted to the XLII series. ##STR38## Where R is D (XLIId), E (XLIIe) or F (XLIIf)

In reactions similar to the above conversion of XII to the XI series, XLI is converted to the XXXIX series. ##STR39## Where R is A (XXXIXa), B (XXXIXb) or G (XXXIXc).

In reactions similar to the above conversion of VII to XIV and XVIIg, XXXVI is converted to XLIV and XLVIg. ##STR40##

In reactions similar to the above conversion of IX to XIXh, XXXVIII is converted to XLVIIIh. ##STR41##

In reactions similar to the above conversion of VII to XXII series, XXXVI is converted to the LI series. ##STR42## 1,5,9,14,18,22-hexabenzyloxycarbonyl-1,22-diamino-11-[N(5'-carboxypentyl)carbonylmethoxy-5,9,14,18-tetraazadocosane ##STR43## Where R is: Pep3 (SEQ ID NO:33) (LIi), Pep4 (SEQ ID NO:34) (LIj), Pep5 (SEQ ID NO:35) (LIk) or Pep6 (SEQ ID NO:36) (LIl)

Intercalating Hexacationic DNA Binding Templates

A schematic flow chart for the synthesis of these compounds is shown in FIG. 8. The chemical pathway of synthesis is shown below.

Combine 2 mmol III (resolved S enantiomer), 4 mmol 4-pyrrolidinopyridine and 4.1 mmol benzyloxycarbonyl anhydride in 40 ml anhydrous benzene and stir overnight at room temperature under N₂. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product LII as an amorphous solid. ##STR44##

Add 4 ml of dry benzene to 1 mmol K₂ CO₃ and 2 mmol 18-crown-6 and stir for 20 min. Add 2 mmol LIII in 4 ml of dry benzene, followed by 2 mmol methyl bromoacetate in 2 ml benzene. After 4 hr, add 25 ml of water and extract with 3 portions of 25 ml benzene. Remove the solvent in vacuo to obtain the product: ##STR45##

Dissolve 5 mmol LIV in 10 ml dry pyridine containing 0.1 mmol dimethylaminopyridine and 15 mmol triethylamine. Add dropwise 11 mmol 6,9-dichloro-2-methoxyacridine on any DNA binding dye that reacts specifically with amino group in 10 ml dry pyridine to the stirred solution. Stir for over 1 hr at room temperature. The solvents are removed in vacuo, and the mixture is redissolved in acetonitrile for solid phase extraction on phenyl-silica and elution with a linear gradient of acetonitrile to 50% in hexane. The appropriate fractions were pooled and the solvent evaporated to obtain the product as an amorphous solid. ##STR46##

Dissolve 2 mmol LV in 10 ml ethanol containing 2 mmol potassium hydroxide. After overnight at room temperature, the solution is transferred to a separatory funnel, to which 2 mmol of HCl, 5 ml of water and 25 ml of benzene is added. After extraction with 3 additional portions of benzene, the combined organic phase is taken to dryness in vacuo. Dissolve in dry DMF for standard solid phase peptide synthesis. ##STR47## 1,12-N,N'-di(2-methoxy-6-chloroacridinyl)amino-4,9-di-t-butyoxycarbonyl-6-carboxymethoxy-4,9-diaza-dodecane

In reactions similar to the conversions in Example 2, the following products are obtained. One skilled in the art will recognize that the reaction conditions are similar, but that the starting material and end product will be different. ##STR48## Where R is A (LVIIa), B (LVIIb), G (LVIIc) or H (LVIII). ##STR49## Where R is A (LIXa), B (LIXb), G (LIXc) or H (LX). ##STR50## Where R is D (LXIId), E (LXIIe) or F (LXIIf). ##STR51## Where R is A (LIXa) B (LIXb) or G (LIXc). ##STR52## Where R is Pep3 (SEQ ID NO:33) (LXXIi), Pep4 (SEQ ID NO:34) (LXXIj), Pep5 (SEQ ID NO:35) (LXXIk) or Pep6 (SEQ ID NO:36) (LXXIL)

Further Intercalating Hexacationic DNA Binding Template

A schematic flow chart for the synthesis of these compounds is shown in FIG. 9. The chemical pathway of synthesis is shown below.

In reactions similar to the conversions disclosed above, the following products are obtained. One skilled in the art will recognize that the reaction conditions are similar, but that the starting material and end products will be different.

After derivatization of the E-N-lys with succinic anhydride, the following ligands are coupled to the resin bound protected peptide.

    H.sub.2 N--CH.sub.2 CH.sub.2 --S--S--CH.sub.2 CH.sub.2 --NH--R

Where R is A, B, G or H.

deprotection and release from the resin yields: ##STR53## Where R is A (LXXIIa), B (LXXIIb), G (LXXIIc) or H (LXXIII). ##STR54## Where R is D (LXXVd), E (LXXVe) or F (LXXVf). ##STR55## The addition of the nuclear localization sequence yields: ##STR56## where R is Pep7 (SEQ ID NO:37) (LXXIXi), Pep8 (SEQ ID NO:38) (LXXIXj), Pep9 (SEQ ID NO:39), (LXXIXk) or Pep10 (SEQ ID NO:40) (LXXIXl) ##STR57## where R is Pep3 (SEQ ID NO:33) (LXXXi), Pep4 (SEQ ID NO:34) (LXXXj), Pep5 (SEQ ID NO:35) (LXXXk) or Pep6 (SEQ ID NO:36) (LXXX1).

Dimeric Octacationic DNA Binding Templates

A schematic flow chart for the synthesis of these compounds is shown in FIG. 10. The chemical pathway of synthesis is shown below.

Combine 2 mmol LIV, 1 mmol 4-pyrrolidinopyridine and 1.0 mmol benzyloxycarbonyl anhydride in 40 ml anhydrous benzene and stir overnight at room temperature under N₂. Separate the product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate, 0 to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the produce LXXXI as an amorphous solid. ##STR58##

Combine 2 mmol LXXXI, 2 mmol 4-pyrrolidinopyridine and 3 mmol bis-(3-carboxyethyl)dithiol in 40 ml anhydrous benzene and stir overnight at room temperature under N₂. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product LXXXII as an amorphous solid. ##STR59##

Dissolve 2 mmol LXXXII in 2 ml dry DMF, add 4.0 mmol 1-ethyl-3-[3-(dimethyl-amino)propyl)carbodiimide and stir 2 hr, then add 2.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr at room temperature, then combine with 2 mmol 3-[(3"-N-t-BOC-aminopropyl)-4'-N-t-BOC-aminobutyl]-N-t-BOC-aminopropylamine in 21 ml dry DMF. After stirring overnight at room temperature, remove the solvent in vacuo, separate the product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate, 0 to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product LXXXIII as an amorphous solid. ##STR60##

Dissolve 2 mmol LXXXIII in 10 ml ethanol containing 2 mmol potassium hydroxide. After overnight at room temperature, the solution is transferred to a separatory funnel, to which 2 mmol of HCl, 5 ml of water and 25 ml of benzene is added. After extraction with 3 additional portions of benzene, the combined organic phase is taken to dryness in vacuo. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product LXXXIV as an amorphous solid. ##STR61##

Dissolve 1 mmol of LXXXIV in 2 ml dry dimethylformamide, add 3.0 mmol 1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hrs, then add 1.1 mmol n-hydroxysuccinimide and continue stirring for another 6 hrs at room temperature. This solution is added dropwise to 3 mmol of H₂ N--CH₂ CH₂ --NH--R (where R=A, B, G or H) in 20 ml dry dimethylformamide, and stirring continued for an additional 24 hrs. Remove the solvent in vacuo. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate from 0 to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product as an amorphous solid. ##STR62## where R is A (LXXXVa), B (LXXXVb), G (LXXXVC) or H (LXXXVI).

Dissolve LXXXVa, 1 mmol, in 29 ml glacial acetic acid containing 30% HBr and stir overnight at room temperature in the dark under N₂. Add 30 ml diethyl ether to precipitate the product. Wash the product until the odor of acetic acid is gone. Dissolve the solid in oxygen-free 0.1 M NH₄ OH. The solution is applied to an anion exchange resin equilibrated in degassed 0.1 M NH₄ CO₃ containing 20% acetonitrile. The product is separated from unreacted starting materials by a gradient of 20 to 80% acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product: ##STR63## where R is A (LXXXVIIa), B (LXXXVIIb), G (LXXXVIIc) or H (LXXXVIII).

Use the same procedures as for LXXXVa-LXXXVc, except substitute S-t-BOC-mercaptoethylamine for H₂ N--CH₂ CH₂ --SS--CH₂ CH₂ --NH--R. Then use the same procedures as for LXXXVIIa-LXXXVIIc. ##STR64## where R is D (XCId), E (XCIe) or F (XCIf)

Dissolve 1 mmol XC in 2 ml phosphate-buffered saline, pH 7.4, and combine with 1 mmol A', also dissolved in 2 ml phosphate-buffered saline, pH 7.4. Dilute the reaction mixture 20-fold with water, apply to an anion exchange resin equilibrated in degassed 0.1 M NH₄ CO₃ containing 20% acetonitrile. The product XIa is separated from unreacted starting materials by a gradient of 20 to 80% acetonitrile in 0.1 M NH₄ CO₃. The appropriate actions are pooled by lyophilized to obtain the product. ##STR65## where R is A (LXXXVIIa), B (LXXXVIIb) or G (LXXXVIIc).

Dissolve 1 mmol of LXXXIV in 2 ml dry dimethylformamide, add 3.0 mmol 1-ethyl-3-[3-(diamethylamino)propyl) carbodiimide and stir 2 hr, then add 1.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr at room temperature. This solution is added dropwise to 5 mmol of 1,6-diaminohexane in 20 ml dry diamethylformamide, and stirring continued for an additional 24 hours. Remove the solvent in vacuo. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate from 0 to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product as an amorphous solid. ##STR66##

Combine 1 mmol XCII in dry 10 ml benzene with 1.1 mmol succinimidyl 3(2-pyridylthio) propionate, stir for 2 hr at room temperature, and then remove the solvent in vacuo. ##STR67##

Dissolve XCIII, 1 mmol, in 20 ml glacial acetic acid containing 30% HBr and stir overnight at room temperature in the dark under N₂. Add 30 ml diethyl ether to precipitate the product. Wash the product until the odor of acetate acid is gone. Dissolve the solid in oxygen-free 0.1 M NH₄ CO₃. The solution is applied to an anion exchange resin equilibrated in degassed 0.1 M NH₄ CO₃ containing 20% acetonitrile. The product is separated from unreacted starting materials by a gradient of 20 to 80% acetonitrile in 0.1 M NH₄ CO₃. The appropriate fractions are pooled and lyophilized to obtain the product. ##STR68##

To a stirred solution of 10 mg of Fab'--SH in 5 ml PBS, pH 7.4, at 4°, add 0.3 ml of 10 mM XCIV in PBS, pH 7.4, dropwise. After 60 min, dialyze against 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hr. ##STR69##

Combine 2 mmol LXXXVI, 2 mmol 4-pyrrolidinopyridine and 3 mmol succinic anhydride in 40 ml anhydrous benzene and stir overnight at room temperature under N₂. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product XCVI as an amorphous solid. ##STR70##

Couple XCVI to the amino terminal of the peptide on the support using standard solid phase peptide methods, cleave from the resin and deprotect, and purify by ion exchange chromatography. ##STR71##

Dissolve 2 mmol LXXXIV in 2 ml dry DMF, add 4.0 mmol 1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hr, then add 2.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr at room temperature, then combine with 2 mmol methyl 6-aminohexanoate in 2 ml dry DMF. After stirring overnight at room temperature, remove the solvent in vacuo, separate the product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate, 0 to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product as an amorphous solid. ##STR72##

Dissolve 2 mmol XCVIII in 10 ml ethanol containing 2 mmol potassium hydroxide. After overnight at room temperature, the solution is transferred to a separatory funnel, to which 2 mmol of HCl, 5 ml of water and 25 ml of benzene is added. After extraction with 3 additional portions of benzene, the combined organic phase is taken to dryness in vacuo. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate to 100% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product as an amorphous solid. ##STR73##

Couple XCIX to the amino terminal of the appropriate peptide on the support using standard solid phase peptide methods, cleave from the resin and deprotect, and purify by ion exchange chromatography. ##STR74## where R is Pep3 (SEQ ID NO:33) (XCXi), Pep4 (SEQ ID NO:34) (XCXj), Pep5 (SEQ ID NO:35) (XCXk) or Pep6 (SEQ ID NO:36)(XCXl).

Further Dimeric Octacationic DNA Binding Templates

A schematic flow chart for the synthesis of these compounds is shown in FIG. 11. The chemical pathways of synthesis are shown below.

After derivatization of the ε-N-Lys or α-N-Tyr with succinic anhydride the following ligands are coupled to the resin bound protective peptide: H₂ N--CH₂ CH₂ --S--S--CH₂ CH₂ --NH--R where R is A, B or G.

Deprotection and release from the resin yields: ##STR75## where R is A (CIa), B (CIb) or G (CIc).

After derivatization of the ε-N Lys with t-BOC--S--(CH₂)₂ --COOH, deprotection and release from the support yields: ##STR76##

To a stirred solution of 10 mg of D or E in 5 ml PBS, pH 7.4, at 4°, add 0.3 ml of 10 mM CII in PBS, pH 7.4, dropwise. After 60 min. dilute the reaction mixture 20-fold with water, apply to a cation exchange column to separate the desired product from unreacted starting material and other products, using a linear gradient formed from equal volumes of water and 2.0 M HCl. The appropriate fractions are pooled and lyophilized to obtain the product. Alternatively, to a stirred solution of 10 mg of F or F' in 5 ml PBS, pH 7.4, at 4° add 0.3 ml of 10 mM CII in PBS, pH 7.4, dropwise. After 60 min, dialyze against 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hr. ##STR77## where R is D (CIIId), E (CIIIe) or F (CIIIf).

After derivatization of the ε-N-succinyl-lys with H₂ N--CH₂ --CH₂ --S--S--CH₂ CH₂ --NH--t-BOC and deblocking, the ligand Pep2 is synthesized on the resin using standard solid phase technique. Deblocking and cleavage from the resin yields: ##STR78##

The resin bound Lys-εNH--CO(CH₂)₅ NH₂ intermediate is coupled with succinimidyl 3(2-pyridyldithio) propionate and then deprotected and cleaved to yield: ##STR79## The addition of the nuclear localization sequence yields: ##STR80## where R is Pep7 (SEQ ID NO:37) (CVIIi), Pep8 (SEQ ID NO:38) (CVIIj), Pep9 (SEQ ID NO:39) (CVIIk) or Pep10 (SEQ ID NO:40) (CVIIl). ##STR81## where R is Pep3 (SEQ ID NO:33) (CVIIIi), Pep4 (SEQ ID NO:34)(CVIIIj), Pep5 (SEQ ID NO:25) (CVIIIk) or Pep6 (SEQ ID NO:36) (CVIIIl).

Octacationic DNA Binding Templates with Dual Ligands

With at least 8 DNA binding templates and at least 12 receptor ligands, there are many possible combinations which can be used. Representative examples include polyamine templates with either a cleavable or non-cleavable spacer joining the templates and oligopeptide templates with either a cleavable or non-cleavable spacer joining the template.

A schematic flow chart for the synthesis of these compounds is shown in FIG. 12. The chemical pathway of synthesis is shown below for polyamine templates:

Final coupling are the same for each peptide as described for the A, A', B, B', G, G' ligands. Reaction conditions for D, E, F and the Fab' are comparable. ##STR82##

In reactions similar to those in Example 7 the following products are found. One skilled in the art will recognize that the starting material and resulting products are different but the reaction is the same. ##STR83## where R is A (CXIa), B (CXIb) or G (CXIc). ##STR84## where R is A (CXIIa), B (CXIIb) or G (CXIIc).

Dissolve 2 mmol CX in 2 ml dry DMF, add 4.0 mmol 1-ethyl-3-[3 (dimethylamino)propyl) carbondiimide and stir 2 hr, then add 2.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr at room temperature, then combine with 2 mmol S(2-aminoethyl) S' (2-pyridyl)-dithiol in 2 ml dry DMF. After stirring overnight at room temperature, remove the solvent in vacuo, separate the product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate, 0 to 50% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product as an amorphous solid. ##STR85##

Dissolve 2 mmol CVIII in 10 ml ethanol containing 20 mmol piperidine. After overnight at room temperature, the solution is transferred to a separatory funnel, to which 50 mmol of HCl, 5 ml of water and 25 ml of benzene is added. After extraction with 3 additional portions of benzene, the combined organic phase is taken to dryness in vacuo. Separate the desired product by solid phase extraction on phenyl-silica and elution with a linear gradient of ethyl acetate to 100% in hexane. Pool the appropriate fractions and evaporate the solvent to obtain the product as an amorphous solid. ##STR86##

Using the same procedures for CXIII, except substituting S-t-BOC-mercaptoethylamine for H₂ N--CH₂ CH₂ --SS--C₅ H₄ N. ##STR87##

The nuclear localization sequences are added. Standard continuous-flow solid phase synthetic methodologies are used to couple the commercially available 5-(N-t-BOC) aminohexanoic acid to the protected peptide on the solid support and the subsequent reaction to give CXVIIa-c, CXVIII and CXIX, as the final products after deprotection and release from the support and chromatographic isolation. ##STR88## Where R is S--CH₂ CH₂ --NH--A, S--CH₂ CH₂ --NHCO--B, S--CH₂ CH₂ --NHCO--L, S--C₅ H₄ N or S-t-BOC ##STR89## Where R is S--CH₂ CH₂ --NH--A (CXVIIa), S--CH₂ CH₂ --NHCO--B (CXVIIb), S--CH₂ CH₂ --NHCO--G (CXVIIc), S--C₅ H₄ N (CXVIII) or SH (CXIX).

To a stirred solution of 10 mg of Fab'-SH in 5 ml PBS, pH 7.4, at 4°, add 0.3 ml of 10 CXVIII in PBS, pH 7.4, dropwise. After 60 min, dialyze against 3 changes of 0.5 L PBS pH 7.4, at 4°, each for 2 hr. Alternatively to a stirred solution of HS--CH₂ CH₂ --CONH-Pep2 (SEQ ID NO:32)-COOH, prepared by standard solid phase peptide methodology, 10 mg in 5 ml PBS, pH 7.4, at 4°, add 0.3 ml of 10 mM CXVIII in PBS, pH 7.4, dropwise. After 60 min, ##STR90## Where R is S-Fab' (CXXg) or S--CH₂ CH₂ --CONH-Pep2 (SEQ ID NO:32)-COOH (CXXh).

Using similar reaction conditions as for preparing CIIId-f ##STR91##

Substitution of succinic anhydride for HOOC--CH₂ CH₂ --S--S--CH₂ CH₂ --COOH₂ --C₆ H₃ (--OCH₃)₂ at the fourth stage of synthesis gives a noncleavable intermediate which is further modified according to the reaction sequences for CXVIIa, CXVIIb, CXXg, CXXh. CXXId, CXXIe and CXXIf to give the following products for gene delivery.

Standard continuous-flow solid phase synthetic methodologies are used to couple succinic anhydride to the protected peptide on the solid support and the subsequent reaction to give CXXIIa-c, and CXXVh, as the final products after deprotection and release from the support and chromatographic isolation. The intermediates corresponding to CXVIII and CXIX, are deprotected, released from the support, chromatographically purified, and reacted with the appropriate intermediates to give CXXVg, CXXVId-f, as described for the CXX and CXXI series. ##STR92## where R is S--CH₂ CH₂ --NH--A (CXXIIa), S--CH₂ CH₂ --NHCO--B (CXXIIb), S--CH₂ CH₂ --NHCO--G (CXXIIc), S-Fab' (CXXVg), S--CH₂ CH₂ --CONH-Pep1 (SEQ ID NO:31)-CONH2 (CXXVh), S--CH₂ CH₂ CO--D (CXXVId) , S--CH₂ CH₂ --CO--E (CXXVIe) or S--CH₂ CH₂ --CONH--F (CXXVIf)

Further Octacationic DNA Binding Templates with Dual Ligands

A schematic flow chart for the synthesis of these compounds is shown in FIG. 13. The chemical pathway of synthesis is shown below.

Final coupling are the same for each peptide as described for the A, A', B, B', G, G' ligands. Reaction conditions for D, E, F and the Fab' are comparable.

For oligopeptide template ##STR93##

Further extension of template yields: ##STR94##

After derivatization of the ε-N-lys with succinic anhydride the ligands (X) are coupled to the resin bound protective peptide.

X where X is H₂ N--CH₂ CH₂ --S--S--C₅ H₄ N, H₂ N--CH₂ CH₂ --S-t-BOC or H₂ N--CH₂ CH₂ --S--S--CH₂ CH₂ --NH--R and

where R is A, B or G.

Although not necessary, it is sometimes desirable to derivatize the amino-terminal tyr with a 2-methoxy-6-chloracridinyl moiety. If not, then deprotection and release from the resin yields: ##STR95## Where R is S--CH₂ CH₂ --NHCO--A (CXXVIIa), S--CH₂ CH₂ --NHCO--B (CXXVIIb), S--CH₂ CH₂ --NHCO--G (CXXVIIc), S--C₅ H₄ N (CXXIX) or SH (CXXX).

CXXX+C₅ H₄ N--S--S--CH₂ CH₂ --CONH--R; where R is D, E or F ##STR96## Where R is D (CXXXId), E (CXXXIe) or F (CXXXIf)

After derivatization of the ε-N-succinyl-lys with H₂ N--CH₂ CH₂ --S--S--CH₂ CH₂ --NH-t-BOC and deblocking the ligand Pep3 (SEQ ID NO:33) is synthesized on the resin using standard solid phase techniques. Deblocking and cleavage from the resin yields: ##STR97##

Substitution of t-BOC--NH--(CH₂)₅ --COOH for FMOC--NH--CH₂ CH₂ --S--S--CH₂ --CH₂ --COOH at the second stage of synthesis gives a noncleavable intermediate, which is further modified according to the reaction sequence for the CXXVII series, CXXXg, CXXXIIIh and the CXXXI series to give the following products for gene delivery. ##STR98## where R is S--CH₂ CH₂ --NH--A (CXXXIVa), S--CH₂ CH₂ --NHCO--B (CXXXIVb), S--CH₂ CH₂ --NHCO--L (CXXXIVc), S-Fab' (CXXXIXg), S--CH₂ CH₂ --CONH-Pep1 (SEQ ID NO:31)-CONH₂ (CXXXVIIIh), S--CH₂ CH₂₋ CO--D (CXXXVIId), S--CH₂ CH₂ --CO--E (CXXXVIIe), S--CH₂ CH₂ --CONH--F (CXXXVIIf).

Oligonucleotides Containing Receptor Ligands

FIG. 14 show a double stranded DNA vector with a single stranded DNA attached to a ligand containing both a plasma membrane and nuclear membrane receptors. Two different functions can be shown for this single stranded pyrimidine deoxyoligonucleotide modified at the 3' and 5' terminal nucleotides with a space molecule derivatized with a ligand for either a plasma membrane receptor or nuclear membrane receptor and/or a nucleotide containing a modified base conjugated with a ligand for either a plasma membrane receptor or a nuclear membrane receptor.

The first function is to target double stranded vectors to specific cells and then to the nucleus of the targeted cell for expression of the vector and/or integration of the vector sequences into the host genome. One to ten copies of the double stranded target sequences either individually or clustered will be inserted in non-coding regions of the vector.

The second function is to deliver therapeutic single stranded DNA for treatment of cancer, infectious disease and cardiovascular disease. Targeting the single stranded DNA to specific cells and then to the nucleus of the targeted cell will form a triplex structure that prevents transcription of the specified genes.

In FIG. 15A is shown single stranded DNA as a DNA-binding template containing a single receptor ligand. C^(m) is the 5-methyl cytosine derivative.

In FIG. 15B is shown single stranded DNA-binding template in which N-(2-ethylamino)glycine replaces the deoxyribose-phosphate backbone of the nucleic acid polymer.

Derivatives and analogs of FIG. 15 are shown in FIG. 15C.

In FIG. 15D is an example of a ligand containing template in the pyrimidine series. With at least 18 binding templates and at least 12 receptor ligands there are many possible combinations for use. It is obvious to one skilled in the art that two different DNA binding templates could be linked 5' (3') to 3' (5') with a dithio bridge so that the single stranded DNA bearing the plasma membrane ligand would disassociate from the double stranded DNA vector. The oligonucleotides are made by conventional solid phase synthesis. The 5' and 3' nucleotides contained in an amino group in lieu of the 5' and 3' hydroxyl moieties, respectively, of the terminal nucleotides. The nucleotide T--Y is 5-(N-[N-{N-ligand-5-aminohexanoyl}-4-aminobutanoyl]-3-aminoallyl]-2'-deoxyuridine moiety. The nucleotide A--Y is 8-[N-[N-ligand-5-aminohexanoyl]-8-aminohexylamino]-2'-deoxyadenosine moiety.

The ligand for SV-40 sequences is shown in FIG. 16. Therapeutic single stranded DNA for the treatment of cancer, infectious disease and cardiovascular disease can be delivered to specific cells and then to the nucleus of the targeted cell where it forms triplex structures that prevent transcription of the specified genes. An initial template will contain two different single stranded DNA templates linked 5' (3') to 3' (5') with a dithio bridge, so that as a result of reduction in the cytoplasm, both the single stranded DNA and the plasma membrane ligand will disassociate from the double stranded DNA vectors as separate molecules. The spacer for the plasma membrane ligand also contains a dithio moiety so that the cellular targeting ligand will be released when the complex is present in the cytoplasm. The oligonucleotides are made by conventional solid phase synthesis. The 5' and 3' nucleotides contain an amino group in lieu of the 5' and 3' hydroxyl moiety, respectively, of the terminal nucleotides. FIG. 17 shows an example of this for the C-myc promoter.

In FIG. 17, the deoxyoligonucleotide strands are synthesized on an automated DNA synthesizer using a solid-phase cyanoethylphosphoramidate method. Commercially available reagents are used to provide a 3' terminal thiol which is reacted further with reacted A after deblocking and release of the oligonucleotide from the support. Dissolve 2 mmol of the protected peptide N-succinyl-Pep5 (SEQ ID NO:35)-CONH₂ released from the peptide support in 2 ml dry DMF, and add 4.0 mmol 1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide. Stir for 2 hr, then add 2.1 mmol N-hydroxysuccinimide. Stir for another 6 hr at room temperature, then couple with the deprotected side chain amino group of 5-(N-[N-{N-ligand-5-aminohexanoyl}-4-aminobutanoyl]-3-aminoallyl)-2'-deoxyuridine moiety on the solid support. The nucleotide is terminated with the commercially available Uni-Link AminoModifier. The terminal Fmoc amino protecting group is removed and reacted with 6,9-dichloro-2-methoxyacridine before the substituted oligonucleotide is cleaved from the support.

Adenovirus Production

Adenovirus (d1312) was grown in 293 cells and purified by double banding on CsCl gradients and then dialyzed against 2× filtered HEPES Buffered Saline (HBS; 150 mM NaCl, 20 mM HEPES, pH 7.3). The concentration of the virus was determined by U.V. spectrophotometric analysis and either stored in 10% glycerol at -20° C. or further modified for DNA complex formation. Adenovirus was thawed and the appropriate amount added directly to hepatocytes for analysis.

Hepatocyte Isolation and Culture

Mouse hepatocytes isolation and culture was by collagenase perfusion technique. Briefly, the mice are anesthetized, the abdomens opened and a cannula is rapiidly inserted into the portal vein for infusion of Earle's balanced salt solution (w/o Mg, w/o Ca) supplemented with 0.5 mM EGTA and 10 mM HEPES pH 7.4. After 5 min, the liver is perfused for 10 to 12 min with regular Earle's balanced salt solution containing 10 mM HEPES pH 7.4, 0.3 mg/ml collagenase, and 0.04 mg/ml soybean trypsin inhibitor. Next, the liver is transferred and dispersed with forceps into a petri-dish containing regular incubation media. The suspension is then filtered through two layers of nylon mesh and the cells are separated from debris by centirfuging and resuspending them 3 times with media. The desired number of hepatocytes are then plated in a Primaria dish (e.g., 3×10⁵ cells/well of a 6 well plate) in regular incubation media.

Cell Lines and Hepatocyte Isolation

The rat embryo fibroblast 208 F cell lines were grown in High glucose DMEM containing 10% heat inactivated hyclone, 1 mM glutamine, 100 μl/ml streptomycin and 100 units/ml penicillin. Mouse hepatocytes were isolated by the collagenase perfusion method and then cultured by methods known in the art. The cultured hepatocytes and liver tissue were tested with X-gal histochemical staining.

Preparation of ASOR and Uptake Studies in Primary Hepatocytes

Orosomucoid was desialylated with neuraminidase to form asialoorosomucoid (ASOR). Residual sialic acid was determined to be less than 5% by the thiobutyric acid assay. The labeling of ASOR was performed by using ³ H-borohydride. Hepatocyte isolation from 10-12 week old C57-B6 mice and 10-12 week old PAH deficient mice was done by collagenase perfusion. The hepatocytes were plated at a density of 3×10⁵ /well in 6 well Primaria plates and grown in 2 ml of Low Glucose complete media (Low Glucose DMEM, 10% fetal calf serum, 10 mM HEPES, 0.5% MEM amino acids, 2 mM glutamine, 100 units/ml penicillin, and 100 pg/ml streptomycin). All uptake studies in hepatocytes were done 2-4 hours after the hepatocytes were plated out, allowing the hepatocytes to attach to the plate. The uptake studies of ³ H-ASOR were performed by incubating the protein with the hepatocytes for the specified period of time after which, the media containing the protein was removed, the cells were washed with PBS (Phosphate Buffered Saline, minus Ca²⁺ and minus Mg⁺), 1 ml of trypsin added to each well and the cells incubated for 10 minutes at room temperature. The trypsinized cells were removed from the plate and pelleted after which the pellet was washed with PBS minus and then the cells were lysed with 0.5 NaOH. Internalized tritium counts were determined from the cell lysates by scintillation counting.

Preparation of ASOR/Poly-L-Lysine/DNA Complexes

Poly-L-lysine (PLL) MW. 20,500, was coupled to ASOR in a 1 to 2.0 ratio by using 1-ethyl-3-(3-dimethylamniopropyl)carbodiimide (EDC) at pH 7.3. The reaction was incubated for 24 hours at room temperature after which it was concentrated and resuspended in 2M Guanidine-HCl, 50 mM HEPES, pH 7.3, and fractionated by gel filtration on a Superose 6 column with a Fast Protein Liquid Chromatography system (FPLC). Once the conjugate was made and purified, fractions from the FPLC were analyzed on an SDS-PAGE gel to determine those fractions that contained modified ASOR (ASOR/PLL conjugate) only. These fractions were then pooled and dialyzed against 150 mM NaCl, 20 mM HEPES, pH 7.3. prior to complex formation. The DNA plasmid CMV/B-gal containing the E. coli β-galactosidase gene under the control of the CMV enhancer and promoter and the DNA plasmid CMV/hPAH containing the human phenylalanine hydroxylase cDNA under the control of the CMV enhancer and promoter were used as reporter genes. All plasmids were isolated and purified by double CsCl banding. Conjugate/DNA complexes were prepared by diluting the conjugate in 150 μl of HBS (150 mM NaCl per 20 mM HEPES, pH 7.3) and diluting 6 μl of DNA, in 350 μl of HBS. The diluted DNA was added directly to the diluted conjugate while mixing. The reaction was allowed to incubate at room temperature for 30 minutes before analysis. Immediately following the incubation, all complexes were analyzed on 0.8% agarose gels and electrophoresed in TBE, or added directly to hepatocytes for uptake analysis.

Analysis of Complex Uptake and Expression in Primary Hepatocytes

Before adding the complex to the hepatocytes, the complete media was removed and replaced with 1 ml of Low Glucose DMEM containing, 5 mM Ca²⁺ and 2% fetal calf serum. Three micrograms of DNA in complex form was then added to the hepatocytes, followed by the immediate addition of the appropriate amount of adenovirus stock. After a 2 hour incubation at 37° C., 1.5 ml of complete media was added to the hepatocytes and the incubation continued for 24 hours at 37° C. The analysis of β-galactosidase (β-gal) activity was done by staining the cells, using X-gal as a substrate. To quantify the actual amount of β-galactosidase produced, ONPG was used as a substrate with aliquots of cell extracts. Phenylalanine hydroxylase activity in cells extracts was measured as the percent conversion of phenylalanine to tyrosine from cell extracts. Protein concentration in cell extracts was determined by the BCA Micro-Protein assay.

Receptor-Mediated Uptake of ASOR in Primary Hepatocytes

The target tissue for this DNA delivery system is the liver, with the specific delivery directed to hepatocytes. A hepatocyte has approximately 500,000 ASOR receptors, compared to HepG2 cells which contain approximately half the number of ASOR receptors. To determine if the desialylated orosomucoid could be internalized by primary hepatocytes in a receptor-mediated process, a dose-response curve was performed by incubating increasing amounts of ³ H-ASOR with 3×10⁵ hepatocytes. After a 2 hour incubation, the cells were isolated and the internalized ³ H counts were measured. The dose response curve shows that maximal uptake of ³ H-ASOR is 6.5 ng of ASOR/3×10⁵ hepatocytes, which is equivalent to 3.2×10⁵ molecules/cell. The dose response curve is similar for HepG2 cells, but because of the lower number of ASOR receptors, the maximal ASOR uptake is 1.5×10⁵ molecules/cell. No uptake of ASOR occurs in NIH3T3 cells as this cell line contains no ASOR receptors.

To determine the kinetics of ASOR uptake in cultured hepatocytes, a time course analysis was performed. Tritium labeled ASOR (2 μg) was incubated with 3×10⁵ hepatocytes and time points were taken at 0, 30, 50, 90, 120, and 240 minutes after addition of the ³ H-ASOR to the hepatocytes. The time course analysis shows that the internalized amount of ³ H-ASOR increases linearly with time for up to 1 hour and reaches saturation level at 2 hours from the initial incubation with the ³ H-ASOR. When the same analysis is done with HepG2 cells, the internalized amount of ³ H-ASOR increases at lower levels and does not reach saturation as seen in primary hepatocytes.

DNA Delivery to Hepatocytes By Receptor-Mediated Endocytosis

The ASOR was conjugated to poly-L-lysine with the water soluble carbodiimide EDC and after purification, increasing concentrations of the conjugate were incubated with DNA. The extent of ASOR/PLL/DNA complex formation was determined by agarose gel electrophoresis. Based on charge neutralization, as seen by the reduction of electrophoretic mobility of the DNA, interaction between the ASOR/PLL conjugate and DNA started at a molar ratio of 10 to 1. The DNA is completely retarded at molar ratios of 100 to 1 and greater. To assess if the complexes formed were capable of being internalized and also allowed expression of the DNA, the complexes were incubated with primary hepatocytes for 24 hours, after which time, the hepatocytes were analyzed for β-galactosidase activity. The results of X-gal staining showed few or no blue hepatocytes.

To increase the efficiency of DNA delivery and expression in the cells, the replication defective adenovirus d1312 was incorporated into the analysis. The complex was incubated with 3×10⁵ hepatocytes and with increasing adenoviral titer ranging from zero to 3×10¹⁰ viral particles. The results of the analysis show that the adenovirus alone or the complex alone do not produce any blue staining cells, but hepatocytes can be quantitatively stained when 3×10⁹ adenoviral particles are used. When the hepatocytes are incubated with both the complex and increasing titers of adenovirus, there is a correlative increase in the number of blue cells after X-gal staining. To determine quantitatively the amount of β-galactosidase being produced, ONPG analysis was done on cellular extracts from the hepatocytes that had been incubated with complex only, adenovirus only, or with the complex and increasing titers of adenovirus. The analysis shows that 0.006 units per mg of protein of β-galactosidase activity occurs when the complex alone is used and this activity increases to 6.4 units per mg of protein when 3×10⁹. adenoviral particles are also included. This represents a 1000-fold increase in activity.

To determine if this delivery was specifically through the ASOR receptor, hepatocytes were incubated with the ASOR/DNA complex and 3×10⁹ adenoviral particles in the presence of increasing concentrations of free ASOR. The analysis shows that the delivery of the DNA by the complex in the presence of adenovirus is competable by excess ASOR, verifying that the majority of DNA uptake occurs specifically through the ASOR receptor.

Expression of Human Phenylalanine Hydroxylase After Receptor-Mediated Gene Delivery to Deficient Hepatocytes

To determine if the system could be used to reconstitute enzymatic activities in PKU, a plasmid containing the human phenylalanine hydroxylase (PAH) cDNA under the control of the CMV enhancer/promoter was used for complex formation. A PAH-deficient mouse strain has been developed and the hepatic enzyme level is only 3% of normal. Hepatocytes isolated from PAH deficient mice were incubated with complex made with a ratio of ASOR to DNA of 250 to 1. This complex shows maximal hPAH expression over a 24 hour period in hepatocyte extracts that have been incubated with 3 μg of DNA in complex form and 3×10⁹ adenoviral particles. After 24 hours, analysis of the extract from these hepatocytes resulted in the conversion of 2.2% of the phenylalanine substrate to tyrosine, as compared to 0.3% conversion with extracts from the untreated PAH-deficient mouse hepatocytes. When the deficient hepatocytes are incubated with 15 μg and 30 μg of DNA in complex form, the amount of phenylalanine conversion increases to 20% and 26% respectively, which is comparable to the activity that occurs in extracts from normal mouse hepatocytes.

To show that the presence of hPAH activity was due to the DNA/Protein complex and not due to non-specific uptake of the increased level of DNA, the hepatocytes were incubated with 3×10⁹ adenoviral particles along with 30 μg of free CMV/hPAH DNA. The results of the analysis showed that no increase in activity occurs over the activity in the PAH deficient hepatocytes with only a 0.3% conversion of phenylalanine to tyrosine, confirming that the uptake occurs specifically through the presence of the DNA/Protein complex.

Receptor-Mediated Uptake of ASOR/Spermine/DNA Complexes in Primary Hepatocytes

ASOR and spermine complexes were coupled as described above using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The conjugate is purified and fractions from the FPLC analyzed on SDS-PAGE gel for fractions containing modified ASOR (ASOR/spermine). Fractions are then pooled and dialyzed prior to complex formation. The DNA plasmid CMV/B-gal containing the E. coli β-galactosidase gene under the control of the CMV enhancer and promoter and the DNA plasmid CMV/hPAH containing the human phenylalanine hydroxylase cDNA under the control of the CMV enhancer and promoter were used as reporter genes. All plasmids were isolated and purified by double CsCl banding. Conjugate/DNA complexes were prepared by diluting both the conjugate and DNA in HBS (150 mM NaCl per 20 mM HEPES, pH 7.3). The diluted DNA was added directly to the diluted conjugate while mixing. The reaction was allowed to incubate at room temperature for 30 minutes before analysis. Immediately following the incubation, all complexes were analyzed on agarose gels and electrophoresed in TBE, or added directly to hepatocytes for uptake analysis.

Before adding the complex to the hepatocytes, the complete media was removed from the cells and replaced with 1 ml of Low Glucose DMEM containing, 5 mM Ca²⁺ and 2% fetal calf serum. DNA in complex form was then added to the hepatocytes, followed by the immediate addition of the appropriate amount of adenovirus stock. After a 2 hour incubation at 37° C., 1.5 ml of complete media was added to the hepatocytes and the incubation continued for 24 hours at 37° C. The analysis of β-galactosidase (β-gal) activity was done by staining the cells, using X-gal as a substrate. To quantify the actual amount of β-galactosidase produced, ONPG was used as a substrate with aliquots of cell extracts. Phenylalanine hydroxylase activity in cells extracts was measured as the percent conversion of phenylalanine to tyrosine from cell extracts. Protein concentration in cell extracts was determined by the BCA Micro-Protein assay.

As noted above, the complexes were incubated with primary hepatocytes for 24 hours, after which time, the hepatocytes were analyzed for β-galactosidase activity. The results of X-gal staining showed few or no blue hepatocytes. The replication defective adenovirus d1312 was incorporated into the analysis to increase the efficiency of DNA delivery and expression in the cells. The complex was incubated with hepatocytes and with increasing adenoviral titer.

When the hepatocytes are incubated with both the complex and increasing titers of adenovirus, there is a correlative increase in the number of blue cells after X-gal staining. To determine quantitatively the amount of β-galactosidase being produced, ONPG analysis was done on cellular extracts from the hepatocytes that had been incubated with complex only, adenovirus only, or with the complex and increasing titers of adenovirus. In addition, expression of hPAH after receptor-mediated gene delivery to deficient hepatocytes was determined as discussed above.

Preparation and Cellular Uptake of ASOR/Nuclear Ligand/DNA Complexes

The nuclear ligand peptide GYGPPKKKRKVEAPYKA(K)₄₀ WK (SEQ ID NO:60) was coupled to PLL to form the nuclear binding molecule by the same procedures as described above. This peptide contains a tyrosine for incorporation or ¹²⁵ I to quantify binding parameters and to determine stiochiometry of the DNA complex. Binding of the peptide to DNA quenches tryptophan fluourescence and allows the kinetics and thermodynamics of complex formation to be determined. The function of the EAP sequence is to extend the nuclear localization sequence at right angles to the PLL backbone. The peptide is homogeneous by reversed phase HPLC and has the expected molecular weight, determined by fast atom bombardment mass spectroscopy.

ASOR-PLL complexes were prepared as described above. The DNA plasmid CMV/B-gal containing the E. coli β-galactosidase gene under the control of the CMV enhancer and promoter and the DNA plasmid CMV/hPAH containing the human phenylalanine hydroxylase cDNA under the control of the CMV enhancer and promoter were used as reporter genes. All plasmids were isolated and purified by double CsCl banding. ASOR/nuclear ligand/DNA complexes were prepared by diluting the ASOR-PLL and nuclear ligand-PLL conjugates in HBS (150 mM NaCl per 20 mM HEPES, pH 7.3) and diluting the DNA in HBS. The diluted DNA was added directly to the diluted conjugate while mixing. The reaction was allowed to incubate at room temperature for 30 minutes before analysis. Immediately following the incubation, all complexes were analyzed on agarose gels and electrophoresed in TBE, or added directly to hepatocytes for uptake analysis.

Before adding the complex to the hepatocytes, the complete media was removed and replaced with 1 ml of Low Glucose DMEM containing, 5 mM Ca²⁺ and 2% fetal calf serum. DNA in complex form was then added to the hepatocytes, followed by the immediate addition of the appropriate amount of adenovirus stock. After a 2 hour incubation at 37° C., 1.5 ml of complete media was added to the hepatocytes and the incubation continued for 24 hours at 37° C. The analysis of β-galactosidase (β-gal) activity was done by staining the cells, using X-gal as a substrate. To quantify the actual amount of β-galactosidase produced, ONPG was used as a substrate with aliquots of cell extracts. Phenylalanine hydroxylase activity in cells extracts was measured as the percent conversion of phenylalanine to tyrosine from cell extracts. Protein concentration in cell extracts was determined by the BCA Micro-Protein assay.

As noted above, to assess if the complexes formed were capable of being internalized and also allowed expression of the DNA, the complexes were incubated with primary hepatocytes for 24 hours, after which time, the hepatocytes were analyzed for β-galactosidase activity. The results of X-gal staining showed few or no blue hepatocytes.

To increase the efficiency of DNA delivery and expression in the cells, the replication defective adenovirus d1312 was incorporated into the analysis. The complex was incubated with hepatocytes and with increasing adenoviral titers. The results of the analysis show that the adenovirus alone or the complex alone do not produce any blue staining cells, but hepatocytes can be quantitatively stained when adenoviral particles are used. When the hepatocytes are incubated with both the complex and increasing titers of adenovirus, there is a correlative increase in the number of blue cells after X-gal staining. To determine quantitatively the amount of β-galactosidase being produced, ONPG analysis was done on cellular extracts from the hepatocytes that had been incubated with complex only, adenovirus only, or with the complex and increasing titers of adenovirus.

Delivery of DNA into the nucleus is determined by using radiolabeled amino acids. The nuclear ligand is labeled with C14 by procedures well known in the art. The nuclear fraction is then isolated by separation techniques known in the art. Radioactivity levels are measured to determine delivery of DNA to the nucleus.

Potocytosis Mediated DNA Delivery

The ability of DNA/folate/adenovirus complexes were analyzed for delivery of DNA directly into the cytosol of cells. Folate was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in dimethylsulfoxide and was then coupled under hydrous conditions to poly-L-lysine (PLL) under procedures as described above. Adenovirus was attached to PLL with the help of EDC under conditions which inactivates the adenovirus binding domain for its receptor, but leaves the endosomal lysis domain intact. The DNA/folate/adenovirus complexes were used to deliver the E. coli β-galactosidase gene into human epidermoid carcinoma cell line (KB). Twenty-four hours after addition of the complex to the cells, histological staining showed that 20-30% of the cells could be positively stained with X-GAL.

In addition to DNA, other macromolecules including RNA, proteins, lipids and carbohydrates can also be delivered into the cytosol using this delivery system. Folate can also be exchanged by other ligands that are taken up into the cells by caveolae. Other water soluble molecules are taken up into cells via this mechanism as well. The adenovirus can also be replaced by other endosomal or potosomal lysis agents, including but not limited to, viruses, bacteria, proteins, peptides and lipids.

Efficient Non-Viral DNA Delivery With The Endosomal Lysis Agent Listeriolysin

A nucleic acid transporter was formed using the above-described methods. Listeriolysin are only a part of the toxin, harboring the active site, was coupled to PLL. In addition, asialoglycoprotein was coupled to PLL as described above. The DNA/ASOR/LIS complexes were then tested in mammalian cells in vitro and in vivo. DNA was delivered directly into the cells through receptor-mediated endocytosis and was capable of escaping the endosome due to the listeriolysin. Gene expression was elevated in the cytoplasm and the nucleus as compared to use of a DNA/ASOR complex without the use of listeriolysin. As discussed above, the hepatocytes express specifically the asialoglycoprotein receptor which recognizes the asialooromucoid protein used in the DNA complex described herein. The asialooromucoid protein is delivered to the cell interior via receptor-mediated endocytosis.

The listeriolysin toxin forms pores in the endosomal membrane. The diameter of the pores are between 50-100 nanometers (nm), which is large enough for macromolecules to pass through. The pH optimum for listeriolysin is between pH 5.5-7.0. The listeriolysin is membranolytic active between these pHs. The harboring active site which can be used with the above-described procedure, is located in the C-terminal region of listeriolysin. Although the above efficiency of DNA delivery is tested in hepatocytes, listeriolysin can be coupled with other ligands to form nucleic acid complexes and used for nucleic acid delivery to other cell types, depending only upon the ligand being used. Furthermore, all similar microbial toxins and their active subfragments can be incorporated into the nucleic acid complexes for endosomal escape.

Preparation of Folate Labeled Conjugates and DNA/Folate Complexes

Folic acid was dissolved in dimethyl sulfoxide and incubated with an 10 fold excess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for 30 min at room temperature. A 30-fold excess of the activated vitamin was added to ¹²⁵ I-bovine serum albumin (BSA) (89 kBq/μg; NEN) in phosphate buffer and incubated for 4 hours at room temperature. The reaction was quenched with a 50-fold excess of ethanolamine and free folate was separated from the ¹²⁵ I-BSA-folate conjugate by passing the reaction mixture over a Sephadex G-25 column equilibrated with phosphate buffered saline, pH 7.4. To estimate the number of folate molecules coupled to BSA under these conditions the same protocol was used with the exception that ³ H-folic acid (2.01 Gbq/mmol; NEN) was used with unlabeled BSA. A similar protocol was used to couple folate to poly-L-lysine (M_(r) -20,500) with a molar ratio of activated vitamin to poly-L-lysine of 2 to 1. After purification the folate/poly-L-lysine conjugate was dialyzed against 150 mM NaCl, 20 mM Hepes, pH 7.3 (HBS).

The DNA plasmid CMV/β-gal containing the E. coli β-galactosidase gene under the control of the early CMV enhancer and promoter was used as a reporter gene. DNA/folate complexes were prepared by diluting the folate/PLL conjugate in 150 μl HBS to which 6 μg DNA (in 350 μl HBS) was added. After 30 minutes of incubation at room temperature the complexes were analyzed on 0.8% agarose gels or added directly to cells for further analysis.

Uptake of ³ H-Folate and Folate Conjugated ¹²⁵ I-BSA Into Cells

KB, Hela, Caco-2, SW620 and SKOV-3 cells were purchased from the American Type Tissue Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 units/ml) streptomycin (100 μg/ml), and 2 mM L-glutamine. For the culture of Caco-2 cells nonessential amino acids were added. The cells were grown for two passages in folate deficient DMEM containing the above-mentioned supplements 8 to 10 days before each experiment. Normal DMEM has a folate concentration 1000 fold above in vivo levels. The supplemented folate deficient DMEM has sufficient folate to sustain cell growth.

Cells were incubated for up to 2 hours with 25 nM ³ H-folic acid. At the indicated time points the cells were washed once with ice cold PBS, followed by a 30 sec wash with ice cold acid saline (0.15 M NaCl, adjusted to pH 3.0 with glacial acetic acid) to remove surface bound folate and then harvested by scraping in ice cold PBS. The cells were pelleted, washed with PBS, and then dissolved in 0.1 M NaOH. One-half was used for protein determination (BCA-Protein assay) and the other half was counted in a scintillation counter. For ¹²⁵ I-BSA-folate uptake the KB cells were incubated with 1-10 μg ¹²⁵ I-BSA-folate/ml for 2 hours. Cells were harvested to determine internalized radioactivity using the protocol described for folic acid. For competition a 100 fold molar excess of folate was added prior to incubation with the DNA complexes.

Analysis of DNA Complex Uptake in KB, Hela, Caco-2, SW620, and SKOV-3 cells

The cells were harvested 24 hours prior to each experiment and 2×10⁵ cells per well were plated in a 6 well plate, to give about 3-4×10⁵ cells per well on the day of the experiment. Before adding the complexes to the cells the media was replaced by 1 ml of folate deficient DMEM without supplements. 3 μg of DNA in the form of DNA/folate complexes were incubated with the cells for 2 hours and after addition of 2 ml of regular folate deficient media the incubation was continued for 24 hours. For experiments using adenovirus, 3-4×10⁸ viral particles were added immediately after the addition of the DNA/folate complexes to the media. The analysis of β-galactosidase activity was done by using X-gal as a substrate for staining and for quantification of the β-galactosidase activity o-nitrophenylgalactose (ONPG) was used. For competition experiments, a 100 fold molar excess of folate was added prior to incubation with the complexes.

Uptake of Folate and Folate Conjugated BSA in KB Cells

Folate uptake studies were conducted with a physiological concentration of extracellular folate, ranging between 5 to 50 nM. To show that KB cells internalize folic acid, cells were incubated with 25 nM ³ H-folic acid, with samples taken at 0, 15, 30, 60, 90, and 120 minutes. The cells were isolated to measure the internalized ³ H-radioactivity. Within 120 minutes of incubation the uptake of ³ H-folic acid reached saturation and a maximum of 1.5 pmol folic acid/10⁶ cells became internalized (FIG. 30).

To confirm that conjugation of folic acid to a macromolecule does not impair recognition of folate by its receptor, folic acid was covalently coupled to ¹²⁵ I-BSA with the water soluble carbodiimide EDC. Under these coupling conditions an average of 3 folate molecules were conjugated to BSA as based on ³ H-folate content. KB cells were incubated for 120 minutes with increasing concentrations of ¹²⁵ I-BSA-folate and after cell isolation internalized ¹²⁵ I-radioactivity was measured (FIG. 31). The uptake of ¹²⁵ I-BSA-folate was saturable at a BSA concentration between 2 and 4 μg/ml (FIG. 31; closed squares). To show that the BSA uptake is specific for folic acid, the experiment was repeated in the presence of a 100 fold molar excess of folate. Under these conditions more than 90% of the cellular uptake of ¹²⁵ I-BSA-folate was inhibited (FIG. 31; open squares).

Delivery of DNA/Folate Complexes in KB Cells

Folic acid was conjugated to poly-L-lysine as described for BSA to give folate/poly-L-lysine conjugates containing 1-2 folates per poly-L-lysine (PLL) molecule. Increasing concentrations of the folate/PLL conjugate were incubated with DNA and the extent of DNA/folate/PLL complex formation was determined by agarose gel electrophoresis. Based on charge neutralization, as shown by reduction of the electrophoretic mobility of the DNA, the interaction between folate/PLL conjugate and DNA could be detected at a molar ratio of 10 to 1. The movement of the DNA was completely retarded at a molar PLL/DNA ratio of greater 100 to 1. To assess if these complexes could be internalized to allow DNA expression in KB cells, the E. coli β-galactosidase gene was used as a reporter gene. Twenty-four hours after addition of the complex to the cells histological staining showed that less than 0.1% of the cells were positively stained with X-gal. To determine quantitatively the β-galactosidase gene expression ONPG analysis was done on cellular extracts, showing less than 0.005 units β-galactosidase activity per mg protein (FIG. 32).

To determine if adenovirus enhanced gene delivery via the folate receptor internalized through caveolae, replication-defective adenovirus d1312 was incorporated in the protocol. KB cells (4×10⁵) were incubated with DNA/folate complexes in the presence of 4×10⁸ viral particles and twenty-four hours later the cells were analyzed for β-galactosidase expression. With X-gal staining approximately 20 to 30% of the cells were positively stained blue, a dramatic increase in comparison to DNA/folate complexes alone. Quantitative ONPG-analysis on cellular extracts showed 7.0 units of β-galactosidase activity per mg protein, corresponding to an at least 1000 fold increase of activity. The majority of DNA uptake occurred specifically through the folate receptor, since it was competable by a 100 fold excess of free folate (FIG. 32).

Delivery of DNA/Folate Complexes into Tumor Cells Overexpressing the Folate Receptor

To show that folate mediated gene delivery is not restricted to KB cells, other tumor cell lines were selected (Hela, Caco-2, SW 620, and SKOV3 cells), which all overexpress the folate receptor. To determine if the cell lines could take up folic acid, cells were incubated with 25 nM ³ H-folic acid for two hours, after which internalized ³ H-radioactivity was determined. Internalization of folic acid by Hela cells was similar to that of KB cells. By contrast the amount of folic acid internalization in Caco-2, SW620, and SKOV-3 cells was ten to twenty fold lower. This difference in uptake corresponds very well to the differences in mRNA levels of the folate receptor, which have been determined for Hela, Caco-2, and SKOV-3 cells.

Hela, Caco-2, SW620, and SKOV-3 cells were incubated with DNA/folate complexes in the presence or absence of replication-defective adenovirus as described for KB cells. After 24 hours incubation the β-galactosidase activity was determined on cellular extracts by using ONPG analysis. Without replication-defective adenovirus less than 0.005 units β-galactosidase activity per mg protein was detected in all four cell lines. Coincubation with replication-defective adenovirus caused an at least 500 fold increase of β-galactosidase activity only in Hela cells, which was specific for folate, since it was competable by a 100 fold excess of free folate (FIG. 33). By contrast, replication-defective adenovirus did not cause a folate specific enhancement of β-galactosidase activity in Caco-2, SW620, and SKOV-3 cells (FIG. 33).

To exclude the possibility that this result is due to differences in the susceptibility of the cell lines to adenoviral infection, the efficiency of viral infection was determined with the help of a recombinant adenovirus expressing the E. coli β-galactosidase gene. The recombinant adenoviral vector Ad.RSVBgal containing β-galactosidase under the transcriptional control of the RSV-LTR promoter is a replication-deficient human adenovirus. Adenovirus was prepared as described in the art. Briefly, this involved growing 293 cells in 150 mm petri-dishes in Dulbecco's Modified Eagle's Medium with high Glucose (HGDMEN) supplemented with penicillin/streptomycin, glutamine and 10% fetal calf serum. At a confluency of 90%, the media is removed and replication-defective adenovirus at a PFU of 10 per cell is added in 5 ml of media onto the cells. Next, the cells are incubated at 37° C. and every 15 min the plates are gently rocked to redistribute the media over the entire plate. After 1 hour of incubation 15 ml of media is added to each plate and the plates are incubated for 36-48 hr. The cells are harvested and the virus is then purified by two rounds of cesium chloride ultracentrifugation. The purified virus was dialysed in 10 mM Tris HCl (pH 7.4), 1 mM MgCl₂ and stored at 4° C. in CsCl for immediate use. Viral titers were determined by O.D. (particles per ml) and by plaque assay. In most experiments the plaque titer was within one log of the O.D. titer.

4×10⁵ KB, Hela, Caco-2, SW 620, and SKOV-3 cells were incubated with 4×10⁸ viral particles and after 24 h the cells were stained with X-gal. For all five cell lines greater than 95% of the cells stained blue, indicating an equal susceptibility towards adenovirus infection. Therefore, folate mediated DNA delivery does not only require a membrane disruption agent, but is also dependent on high levels of expression of the folate receptor.

Adenovirus Modification and DNA Complex Formation

Freshly isolated adenovirus, as described above, (1.4×10¹¹ particles) was combined with Poly-l-Lysine (PLL) M.W.-20,500, at a concentration of 16 μM, along with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) at a final concentration of either 130 μM (low EDC) or 2600 μM (high EDC) in a final volume of 4 milliliters. After incubation on ice for four hours after which time, the unreacted components were removed by ultra-centrifugation (150,000×g) for 18 hours on a CsCl gradient at a CsCl concentration of 1.35 g/ml. The adenovirus was then either dialyzed against 2M NaCl and then stored at -20° C. in 10% glycerol or stored in 10% glycerol.

The DNA used in complex formation was either the plasmid pCMV/βGal, which contains the beta-galactosidase gene under the control of the CMV enhancer and promoter or the plasmid pCMV/cFIX. This plasmid was constructed as follows. The canine factor IX (cFIX) cDNA (kozak translation sequence constructed as known in the art) was cloned into the Xho I/Cla I sites of the spCMV plasmid. The Xho I/Sal I fragment containing the CMV promoter-enhancer and factor IX cDNA was then cloned into the Xho I site of the N2 retroviral vector in the forward orientation. All plasmid DNA was purified by banding twice in CsCl gradients.

The modified ADV-PLL/DNA complexes were made in a two step procedure. The first step involved the addition of 10 μg of DNA, in 250 μl of HBS, to the ASOR/PLL conjugate in 250 μl, with continuous mixing, followed by incubation at room temperature for 30 minutes. The ASOR/PLL conjugate was synthesized and purified as before. Sufficient ASOR/PLL conjugate to neutralize 75% of the charge on the DNA molecule was used. The second step involved the addition of the modified adenovirus, in 330 μl of HBS, to the DNA/ASOR/PLL mixture, with continuous mixing, followed by incubation for an additional 30 minutes at room temperature. After complex formation, the complexes were either analyzed by electron microscopy or added to the hepatocytes for analysis.

Analysis of Complex Uptake and Expression in Primary Hepatocytes

Hepatocyte isolation from 10-12 week old C57-B6 mice was done by collagenase perfusion. The hepatocytes were plated at a density of 3×10⁵ /well in 6 well Primaria plates and grown in 2 ml of Low Glucose complete media (Low Glucose DMEM, 10% fetal calf serum, 10 mM HEPES, 0.5% MEM amino acids, 2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin). All uptake studies in hepatocytes were done within 2-4 hours after the hepatocytes were plated, allowing for the hepatocytes to attach to the surface. Before adding the DNA complexes to the hepatocytes, the complete media was removed and replaced with 1 ml of Low Glucose incomplete media (Low Glucose DMEM containing, 5 mM Ca⁺² and 2% fetal calf serum). The modified adenovirus/DNA complexes were incubated with cells in incomplete media for 2 hours at 37° C. in a CO₂ incubator, after which the media was removed and replaced with 1 ml of media and the incubation was allowed to continue at 37° C. in a CO₂ incubator.

The analysis of β-galactosidase (β-gal) activity was done by staining the cells, using X-gal as a substrate. The measurement of factor IX levels were analyzed from the supernatants of cultured hepatocytes that were incubated in media for various times. The levels of cFIX in the samples were determined according to a protocol known in the art, utilizing an ELISA based assay. Protein concentration in cell extracts was determined by the BCA Micro-Protein assay.

Receptor Mediated Endocytosis and Adenoviral Mediated Endosomal Release of DNA

The ASOR in the ASOR-PLL/DNA complex functions as a receptor ligand to target the DNA to hepatocytes, while the PLL functions to attach DNA to ASOR through ionic interactions. To achieve efficient cellular delivery of DNA without subsequent lysosomal degradation, the complex can be co-incubated with a replication defective adenovirus. A fraction of the DNA toroids and the adenovirus are internalized in the same endosome, leading to the release of the DNA from the endosome, escaping lysosomal degradation. To reduce the viral titers while enhancing the efficiency of DNA delivery, the adenovirus can be coupled directly to the DNA complex. As a result, the ASOR-PLL/DNA/PLL-ADV complex is co-internalized, either through the adenovirus receptor or the ASOR receptor. After co-internalization, the adenovirus causes endosomal lysis and results in the release of the ASOR/PLL/DNA complex into the cytoplasm.

Ultra-Structure of ADV-PLL/DNA Complexes

The ultra-structure of the ADV-PLL/DNA complexes was determined by electron microscopy. When the ASOR-PLL conjugate is combined with DNA in the proper ratio to achieve charge neutralization on the DNA molecule, a DNA toroid results. After conjugation with PLL, the adenoviral particle retains its natural structure. When the PLL conjugated ADV is incubated with DNA toroids, complexes between DNA toroids and adenoviral particles are formed. When PLL is conjugated to the adenoviral particle at low concentrations of EDC and used in DNA complex formation, the viral particles are linked to DNA toroids in limited numbers. The coupling of a DNA toroid with 1-3 adenovirus particles, usually occurs in the coupling procedure and gives a complex that is less than 200 nm in size. When PLL is conjugated to adenovirus under high EDC concentrations and then used for complex formation, however, multiple viral particles bound to the DNA toroids are frequently observed. These complexes are greater than 200-300 nm in diameter.

Dose Response of Adenovirus/DNA Complexes on Primary Hepatocytes

To analyze the ability of the ADV-PLL/DNA complexes to deliver DNA, primary mouse hepatocytes were used as recipient cells to measure gene expression. Primary hepatocytes (3×10⁵) were incubated with increasing titers of adenovirus, ranging from 0 to 10³ particles in free and conjugated forms. When the cells were incubated with ASOR-PLL/DNA complexes along with free adenovirus, the percentage of cells that express β-gal was 9% when 10³ particles/cell was used. The amount of free virus that is needed to transduce 100% of cells is 10⁴ particles/cell. In contrast, when adenovirus conjugated with PLL at low EDC concentrations was used at 10³ adenoviral particles/cell, the percentage of cells staining positive for beta-gal reached 100%. When adenovirus conjugated with PLL at high EDC concentrations was used, 80% of cells stained blue at 10³ particles/cell.

To determine the percentage of cells that internalized DNA specifically through the ASOR receptor, the same dose response analysis was done as before, but in the presence of a 1000-fold excess of free ASOR. The cells incubated with ASOR-PLL/DNA complexes and free adenovirus showed a decrease in the percentage of cells expressing β-gal to less than 2%, when 10³ particles/cell was used, demonstrating that most of the DNA uptake specifically occurred through the ASOR receptor. Competition of the complex containing adenovirus conjugated with PLL at low EDC concentrations showed a minimal decrease in the percentage of cells staining positive for beta-gal from 100% to about 80% at 10³ adenovirus particles/cell. This result shows that the majority of the DNA uptake of this complex into recipient cells was through the adenovirus receptor. When the adenovirus conjugated with PLL at high EDC concentrations was used however, the percentage of cells staining blue decreases from 80% to less than 30%, when 10³ particles/cell are used, indicating that the majority of the DNA delivered by this complex occurs through the ASOR receptor.

To further determine the basis for uptake by these complexes, the ADV-PLL/DNA complex was made with PLL instead of the ASOR-PLL conjugate and then incubated with cells. This complex, when used to deliver DNA in the presence of free adenovirus resulted in less than 2% of the cells staining positive for Beta-gal. When the low EDC modified adenovirus was used the percentage of blue cells was 80% at 10³ particles/cell. When this was done with the high EDC modified adenovirus, the percentage of blue cells was 37% at 10³ particles/cell. The results were in complete agreement with the competition experiment, suggesting that the residual uptake is due to other interactions between the adenovirus and the cells. Analysis of the cytopathic effect of the complexes on the cells after a 96-hour incubation showed no toxic effects when either the low or high EDC modified ADV-PLL/DNA complexes were used at 10³ particles/cell.

In studies with adenovirus coupled to PLL and included in a DNA/ASOR-PLL complex, the controls confirmed endocytosis by the asialoglycoprotein receptor and not the adenovirus receptor. Uptake of the DNA/ASOR-PLL/adenovirus-PLL complex by hepatocytes gave results identical to those observed with ten-fold higher free virus. With adenovirus/PLL in the DNA complex, little if any cytopathic effect was found after prolonged incubations. SDS gel analysis showed extensive cross-linking of viral proteins by the carbodiimide, therefore, the role of adenovirus is limited to endosome lysis.

Expression of Canine Factor IX in the Primary Mouse Hepatocytes

To quantitatively compare the levels of gene expression achieved with the ADV-PLL/DNA complexes with those achieved with the free adenovirus, the complexes were used to deliver a canine factor IX (cFIX) cDNA into primary mouse hepatocytes (FIG. 34). Adenovirus modified with high EDC concentrations was used to deliver the DNA, since the complexes made with this conjugated adenovirus deliver the DNA primarily through the ASOR receptor. No cFIX activity was observed when the adenovirus alone was used (FIG. 34, Lane 2). When DNA in toroid form was used along with free adenovirus at a titer of 10³ particles/cell, the level of cFIX increased to 0.032 μg/10⁶ cells/24 hours (FIG. 34, lane 4). In contrast, when the conjugated adenovirus was used to deliver DNA at 10³ particles/cell, the levels of cFIX increase to 0.79 μg/10⁶ cells/24 hours (FIG. 34, lane 5). This represents a 25-fold enhancement of cFIX expression over that achieved with the complex and free adenovirus at the same titer.

The above nucleic acid transporter system can be used to deliver cFIX gene to Hemophilia B dogs. Administration of the appropriate concentration of the transporter to obtain a final concentration of 2-5 ng/ml of cFIX in the plasma of treated dogs will result in partial phenotypic correction of hemophilia B. The same can be utilized with the Factor IX gene for hemophiliacs. Likewise, the above transporter system can be used for treating other metabolic disorders such as phenylketonuria and familial hypercholesterolemia.

Use of Perfringolysin O ("PFO") as a Lysis Agent Dose Response of PFO

Sol 8 (muscle cell line) cells were incubated with DNA/Transferrin Perfringolysin O (DNA/TF/PFO) complexes. The DNA plasmid CMV/β-Gal, which contains the E. coli β-galactosidase gene, as described above, was used. The DNA/TF/PFO complex was prepared as described above. TF and PFO were conjugated with PLL using the techniques discussed above. 3 μg DNA/TF/PFO complex was added to 5×10⁵ cells and after 24 hours the cells ONPG-Analysis was done (FIG. 35). PFO1 to PFO6 represent increasing amounts of PFO in the complexes. β-gal activity increased from PFO1 to PFO4. Above PFO4 the specific β-gal activity goes down due to toxic effects of PFO. Toxicity can be avoided by using Listeriolysin O. Comparison of DNA/TF/PFO to DNA/PFO complexes shows that approximately 50% of the β-gal activity is independent of transferrin.

Dose Response of DNA/TF/PFO Complexes

Using the complexes above, 3, 4.5 or 6 μg of the DNA/TF/PFO was added to 5×10⁵ cells and β-gal activity was assayed 24 hours later. An increase of β-gal activity is seen for both complexes. Quantitative analysis of β-gal expression using DNA/TF/PFO4 provided over 25% of the cells to exhibit β-gal activity. DNA/TF/PFO3 exhibited over 20% β-gal activity.

Comparison of PFO and Adenovirus as an Endosomal Lysis Agent

The DNA/TF/PFO complex (PFO4) was compared with adenovirus as to the effect on β-gal expression. The adenovirus is separate from the DNA/TF/PFO complexes. The amount of adenovirus was chosen in such a way that 100% of the cells are blue on X-gal stain. Expression using the adenovirus was greater than tenfold over the use of PFO as a lysis agent.

Liposome Leakage Assay

Liposome membrane activity was measured by testing liposomal leakage. Briefly, a fluorescent dye (calcein) is encapsulated into liposomes at a concentration where the fluorescence of the dye is greatly reduced (self-quenching). When the liposomes are destroyed by the lysis agent, the fluorescent dye leaks out of the liposomes and is diluted in the incubation buffer. This causes a great increase of fluorescence (dequenching) which can be followed in a fluorescence spectrotrophotometer.

Liposomes were incubated with monomeric or dimeric forms of the HA₂ -fusiogenic peptide at a concentration of 0.5 μg/ml in a sodium-citrate buffer with a pH ranging from 4.5 to 7.4. Before and 5 min after the addition of the lysis agent peptides, the fluorescence was determined. The fluorescence corresponding to 100% leakage was determined by complete lysis of the liposomes with a detergent (Triton X-100; final concentration 0.5%) and the values obtained for the monomeric and dimeric form were plotted as percentage leakage (FIGS. 36 and 37). As seen in FIGS. 36 and 37, the dimeric form of the HA₂ -fusiogenic peptide is more potent and its activity is far better controlled by the pH than the activity of the monomeric form.

Treatment of Cardiovascular Disease

In order to treat cardiovascular diseases, it is best to achieve high serum concentrations of High Density Lipoproteins (HDL) and low levels of Low Density Lipoproteins (LDL). This can be accomplished by over expressing a combination of seven proteins in the liver. The proteins are cholesterol-7α-hydroxylase, truncated apolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoprotein A1, LDL receptor, scavenger receptor, molecular variants of each, and combinations thereof.

Nucleic acid transporters containing DNA coding for human cholesterol-7α-hydroxylase, truncated apolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoprotein A1, LDL receptor, scavenger receptor, molecular variants of each, or combinations thereof can be constructed. A full-length cDNA clone of human cholesterol-7α-hydroxylase containing all of the coding sequences is used. The same strategy is used to incorporate truncated apolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoprotein A1, LDL receptor, scavenger receptor, molecular variants of each, and combinations thereof. In some cases, more than one gene, a molecular variant of the given gene, and possibly all seven genes or their molecular variants can be used with the nucleic acid transporters. One example is cholesterol-7α-hydroxylase and apolipoprotein B truncated.

Because of its ease in producing hypercholesterolemia by cholesterol feeding, the heterozygote Watanabe hereditary hyperlipidemic rabbit is a good example for showing the effect of these vectors. The efficacy of this therapeutic approach is monitored by a simple observation of serum cholesterol and triglyceride levels, lipoprotein profiles and apoprotein levels. After intravenous injection of the nucleic acid transporter in experimental animals, (1) the tissue localization of the DNA, (2) the tissue specificity for gene expression, (3) how long the new gene can be expressed and (4) function is determined.

Cell Transformation

One embodiment of the present invention includes cells transformed with nucleic acid associated with the nucleic acid transporter systems described above. Once the cells are transformed, the cells will express the protein, polypeptide or RNA encoded for by the nucleic acid. Cells included but are not limited to liver, muscle and skin. This is not intended to be limiting in any manner.

The nucleic acid which contains the genetic material of interest is positionally and sequentially oriented within the host or vectors such that the nucleic acid can be transcribed into RNA and, when necessary, be translated into proteins or polypeptides in the transformed cells. A variety of proteins and polypeptides can be expressed by the sequence in the nucleic acid cassette in the transformed cells. These proteins or polypeptides which can be expressed include hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, tumor antigens, viral antigens, parasitic antigens and bacterial antigens.

Transformation can be done either by in vivo or ex vivo techniques. One skilled in the art will be familiar with such techniques for transformation. Transformation by ex vivo techniques includes co-transfecting the cells with DNA containing a selectable marker. This selectable marker is used to select those cells which have become transformed. Selectable markers are well known to those who are skilled in the art.

For example, one approach to gene therapy for hepatic diseases is to remove hepatocytes from an affected individual, genetically alter them in vitro, and reimplant them into a receptive locus. The ex vivo approach includes the steps of harvesting hepatocytes, cultivating the hepatocytes, transducing or transfecting the hepatocytes, and introducing the transfected hepatocytes into the affected individual.

The hepatocytes may be obtained in a variety of ways. They may be taken from the individual who is to be later injected with the hepatocytes that have been transformed or they can be collected from other sources, transformed and then injected into the individual of interest.

Once the ex vivo hepatocyte is collected, it may be transformed by contacting the hepatocytes with media containing the nucleic acid transporter and maintaining the cultured hepatocytes in the media for sufficient time and under conditions appropriate for uptake and transformation of the hepatocytes. The hepatocytes may then be introduced into an orthotopic location (the body of the liver or the portal vasculature) or heterotopic locations by injection of cell suspensions into tissues. One skilled in the art will recognize that the cell suspension may contain: salts, buffers or nutrients to maintain viability of the cells; proteins to ensure cell stability; and factors to promote angiogenesis and growth of the implanted cells.

In an alternative method, harvested hepatocytes may be grown ex vivo on a matrix consisting of plastics, fibers or gelatinous materials which may be surgically implanted in an orthotopic or heterotopic location after transduction. This matrix may be impregnated with factors to promote angiogenesis and growth of the implanted cells. Cells can then be reimplanted.

Administration

Administration as used herein refers to the route of introduction of the nucleic acid transporters into the body. Administration includes intravenous, intramuscular, topical, or oral methods of delivery. Administration can be directly to a target tissue or through systemic delivery.

In particular, the present invention can be used for administering nucleic acid for expression of specific nucleic acid sequence in cells. Routes of administration include intramuscular, aerosol, oral, topical, systemic, ocular, intraperitoneal and/or intrathecal. A preferred method of administering nucleic acid transporters is by intraveneous delivery. Another preferred method of administration is by direct injection into the cells.

The special delivery route of any selected vector construct will depend on the particular use for the nucleic acid associated with the nucleic acid transporter. In general, a specific delivery program for each nucleic acid transporter used will focus on uptake with regard to the particular targeted tissue, followed by demonstration of efficacy. Uptake studies will include uptake assays to evaluate cellular uptake of the nucleic acid and expression of the specific nucleic acid of choice. Such assays will also determine the localization of the target nucleic acid after uptake, and establishing the requirements for maintenance of steady-state concentrations of expressed protein. Efficacy and cytotoxicity is then tested. Toxicity will not only include cell viability but also cell function.

Incorporated DNA into transporters, as described herein, which undergo endocytosis increases the range of cell types that will take up foreign genes form the extracellular space.

The chosen method of delivery should result in cytoplasmic accumulation and optimal dosing. The dosage will depend upon the disease and the rough of administration but should be between 1-1000 mg/kg of body weight/day. This level is readily determinable by standard methods. It could be more or less depending on the optimal dosing. The duration of treatment will extend through the course of the disease symptoms, possibly continuously. The number of doses will depend upon disease delivery vehicle and efficacy data from clinical trials.

Establishment of therapeutic levels of DNA within the cell is dependent upon the rate of uptake and degradation. Decreasing the degree of degradation will prolong the intracellular half-life of the DNA.

Direct DNA Delivery to Muscle

The muscular dystrophies are a group of diseases that result in abnormal muscle development, due to many different reasons. These diseases can be treated by using the direct delivery of genes with the nucleic acid transporters of the present invention resulting in the production of normal gene product. Delivery to the muscle using the present invention is done to present genes that produce various antigens for vaccines against a multitude of infections of both viral and parasitic origin. The detrimental effects caused by aging can also be treated using the nucleic acid delivery system described herein. Since the injection of the growth hormone protein promotes growth and proliferation of muscle tissue, the growth hormone gene can be delivered to muscle, resulting in both muscle growth and development, which is decreased during the later portions of the aging process. Genes expressing other growth related factors can be delivered, such as Insulin Like Growth Factor-1 (IGF-1). Furthermore, any number of different genes may be delivered by this method to the muscle tissue. IGF-1 can be used to deliver DNA to muscle, since it undergoes uptake into cells by receptor-mediated endocytosis. This polypeptide is 70 amino acids in length and is a member of the growth promoting polypeptides structurally related to insulin. It is involved in the regulation of tissue growth and cellular differentiation affecting the proliferation and metabolic activities of a wide variety of cell types, since the polypeptide has receptors on many types of tissue. As a result, the nucleic acid transporter delivery system of the present invention utilizes IGF-1 as a ligand for tissue-specific nucleic acid delivery to muscle. The advantage of the IGF-1/nucleic acid delivery system is that the specificity and the efficiency of the delivery is greatly increased due to a great number of cells coming into contact with the ligand/nucleic acid complex with uptake through receptor-mediated endocytosis. Using the nucleic acid described above in the delivery systems of the present invention with the use of specific ligands for the delivery of nucleic acid to muscle cells provides treatment of diseases and abnormalities that affect muscle tissues.

Direct DNA Delivery to Osteogenic Cells

There are many other problems that occur during the aging process, but one major problem is osteoporosis, which is the decrease in overall bone mass and strength. The direct nucleic acid delivery system of the present invention can be used to deliver genes to cells that promote bone growth. The osteoblasts are the main bone forming cell in the body, but there are other cells that are capable of aiding in bone formation. The stromal cells of the bone marrow are the source of stem cells for osteoblasts. The stromal cells differentiate into a population of cells known as Inducible Osteoprogenitor Cells (IOPC), which then under induction of growth factors, differentiate into Determined Osteoprogenitor Cells (DOPC). It is this population of cells that mature directly into bone producing cells. The IOPCs are also found in muscle and soft connective tissues. Another cell involved in the bone formation process is the cartilage-producing cell known as the chondrocyte.

The factor that has been identified to be involved in stimulating the IOPCs to differentiate is known as Bone Morphogenetic Protein (BMP). This 19,000 MW protein was first identified from demineralized bone. Another factor similar to BMP is Cartilage Induction Factor (CIF), which functions to stimulate IOPCs to differentiate also, starting the pathway of cartilage formation, cartilage calcification, vascular invasion, resorption of calcified cartilage, and finally induction of new bone formation. Cartilage Induction Factor has been identified as being homologous to Transforming Growth Factor β.

Since osteoblasts are involved in bone production, genes that enhance osteoblast activity can be delivered directly to these cells. Genes can also be delivered to the IOPCs and the chondrocytes, which can differentiate into osteoblasts, leading to bone formation. BMP and CIF are the ligands that can be used to deliver genes to these cells. Genes delivered to these cells promote bone formation or the proliferation of osteoblasts. The polypeptide, IGF-1 stimulates growth in hypophysectomized rats which could be due to specific uptake of the polypeptide by osteoblasts or by the interaction of the polypeptide with chondrocytes, which result in the formation of osteoblasts. Other specific bone cell and growth factors can be used through the interaction with various cells involved in bone formation to promote osteogenesis.

Non-limiting examples of genes expressing the following growth factors which can be delivered to these cell types are Insulin, Insulin-Like Growth Factor-1, Insulin-Like Growth Factor-2, Epidermal Growth Factor, Transforming Growth Factor α, Transforming Growth Factor β, Platelet Derived Growth Factor, Acidic Fibroblast Growth Factor, Basic Fibroblast Growth Factor, Bone Derived Growth Factors, Bone Morphogenetic Protein, Cartilage Induction Factor, Estradiol, and Growth Hormone. All of these factors have a positive effect on the proliferation of osteoblasts, the related stem cells, and chondrocytes. As a result, BMP or CIF can be used as conjugates to deliver genes that express these growth factors to the target cells by the intravenous injection of the nucleic acid/Protein complexes of the present invention. Using the nucleic acid described above in the delivery systems of the present invention with the use of specific ligands for the delivery of nucleic acid to bone cells provides treatment of diseases and abnormalities that affect bone tissues.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned as well as those inherent therein. The nucleic acid transporter systems along with the methods, procedures, treatments, molecules, specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed here in without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES:       - # 65                                 - -  - - (2) INFORMATION FOR SEQ ID NO:  1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         31 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     double                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  1:                         - - TCCTAGCAAA GGAGGAGACG AAGAAAAATG A        - #                  - #               31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         11 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    Other nucleic acid                   - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "C" stands for 5-methylcytosine           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  2:                         - - TTTCCTCCTC T               - #                  - #                       - #       11                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:  3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         11 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ  ID - #NO:  3:                         - - AAAGGAGGAG A               - #                  - #                       - #       11                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:  4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         23 bases                                           (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     double                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    Genomic cDNA                         - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  4:                         - - TCCAAAAAAG AAGAGAAAGG TAG           - #                  - #                     23                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         17 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    Other nucleic acid                   - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "C" stands for 5-methylcytosine           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  5:                         - - TTTTTTCTTC TCTTTCC             - #                  - #                       - #   17                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:  6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         17 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  6:                         - - AAAAAAGAAG AGAAAGG             - #                  - #                       - #   17                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:  7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         17 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     double                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  7:                         - - AAAGAGAGAG AGAGGGA             - #                  - #                       - #   17                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:  8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         17 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    Other nucleic acid                   - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "C" stands for 5-methylcytosine           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  8:                         - - TTTCTCTCTC TCTCCCT             - #                  - #                       - #   17                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:  9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         17 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  9:                         - - AAAGAGAGAG AGAGGGA             - #                  - #                       - #   17                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:  10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         35 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     double                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  10:                        - - CTACTCGAGA AAGGAGAGAA AAAGGGGCGG TCCCG       - #                  -      #       35                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         21 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    Other nucleic acid                   - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "C" stands for 5-methylcytosine           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  11:                        - - CTCTTTCCTC TCTTTTTCCC C           - #                  - #                       - #21                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:  12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         21 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  12:                        - - GAGAAAGGAG AGAAAAAGGG G           - #                  - #                       - #21                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:  13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         39 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     double                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  13:                        - - CTCTCTAAAA AGGGAGGGGA GGGGAGGGAA AAACTCTCT      - #                       - #    39                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  14:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         27 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    Other nucleic acid                   - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "C" stands for 5-methylcytosine           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  14:                        - - TTTTTCCCTC CCCTCCCCTC CCTTTTT          - #                  - #                  27                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  15:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         27 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  15:                        - - AAAAAGGGAG GGGAGGGGAG GGAAAAA          - #                  - #                  27                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  16:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         11 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    Other nucleic acid                   - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "C" stands for 5-methylcytosine           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  16:                        - - TTTCCTCCTC T               - #                  - #                       - #       11                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:  17:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         10 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  17:                        - - Gly Tyr Ser Thr Pro Gly Arg Lys Lys Arg                                    1               5  - #                 10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  18:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         16 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  18:                        - - His Leu Arg Arg Leu Arg Arg Arg Leu Leu Ar - #g Glu Ala Glu Glu Gly        1               5  - #                 10 - #                 15               - -  - - (2) INFORMATION FOR SEQ ID NO:  19:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         27 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  19:                        - - TGGGGAGGGA GGGGAGGGAG GGGAAGG          - #                  - #                  27                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  20:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         39 base pairs                                      (B) TYPE:     - #           nucleic acid                                       (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  20:                        - - TAGAGGAGGC CGCAGGGGCT GGGCAGGAAG GAGGTGAAT      - #                       - #    39                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  21:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #        21 base pairs                                       (B) TYPE:     - #          nucleic acid                                        (C) STRANDEDNESS:   - #    single                                              (D) TOPOLOGY:    - #       linear                                     - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #       "C" stands for 5-methylcytos       - #ine                                                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 21:                          - - CTCTTTCCTC TCTTTTTCCC C           - #                  - #                       - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:  22:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #        27 base pairs                                       (B) TYPE:     - #          nucleic acid                                        (C) STRANDEDNESS:   - #    single                                              (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    cDNA                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 22:                          - - TGGGGAGGGA GGGGAGGGAG GGGAAGG          - #                  - #                  27                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  23:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         15 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  23:                        - - Pro Asp Glu Val Lys Arg Lys Lys Lys Pro Pr - #o Thr Ser Tyr Gly           1               5   - #                10  - #                15                - -  - - (2) INFORMATION FOR SEQ ID NO:  24:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         12 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  24:                        - - Pro Arg Arg Arg Thr Lys Pro Pro Thr Ser Ty - #r Gly                       1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  25:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         10 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  25:                        - - Arg Lys Lys Arg Gly Pro Thr Ser Tyr Gly                                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  26:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         13 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  26:                        - - Trp Arg Arg Arg Arg Asn Arg Arg Pro Thr Se - #r Tyr Gly                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  27:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         15 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  27:                        - - Gly Tyr Ser Thr Pro Pro Lys Lys Lys Arg Ly - #s Val Glu Asp Pro           1               5   - #                10  - #                15                - -  - - (2) INFORMATION FOR SEQ ID NO:  28:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         12 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  28:                        - - Gly Tyr Ser Thr Pro Pro Lys Thr Arg Arg Ar - #g Pro                       1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  29:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         10 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  29:                        - - Gly Tyr Ser Thr Pro Gly Arg Lys Lys Arg                                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  30:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         13 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  30:                        - - Gly Tyr Ser Thr Pro Arg Arg Asn Arg Arg Ar - #g Arg Trp                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  31:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         16 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  31:                        - - His Leu Arg Arg Leu Arg Arg Arg Leu Leu Ar - #g Glu Ala Glu Glu Gly       1               5   - #                10  - #                15                - -  - - (2) INFORMATION FOR SEQ ID NO:  32:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         15 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  32:                        - - His Leu Arg Arg Leu Arg Arg Arg Leu Leu Ar - #g Glu Ala Glu Glu           1               5   - #                10  - #                15                - -  - - (2) INFORMATION FOR SEQ ID NO:  33:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         15 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  33:                        - - Gly Tyr Ser Thr Pro Pro Lys Lys Lys Arg Ly - #s Val Glu Asp Pro           1               5   - #                10  - #                15                - -  - - (2) INFORMATION FOR SEQ ID NO:  34:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         12 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  34:                        - - Gly Tyr Ser Thr Pro Pro Lys Thr Arg Arg Ar - #g Pro                       1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  35:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         10 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  35:                        - - Gly Tyr Ser Thr Pro Gly Arg Lys Lys Arg                                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  36:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         13 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  36:                        - - Gly Tyr Ser Thr Pro Arg Arg Asn Arg Arg Ar - #g Arg Trp                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  37:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         15 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  37:                        - - Pro Asp Glu Val Lys Arg Lys Lys Lys Pro Pr - #o Thr Ser Tyr Gly           1               5   - #                10  - #                15                - -  - - (2) INFORMATION FOR SEQ ID NO:  38:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         12 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  38:                        - - Pro Arg Arg Arg Thr Lys Pro Pro Thr Ser Ty - #r Gly                       1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  39:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         10 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  39:                        - - Arg Lys Lys Arg Gly Pro Thr Ser Tyr Gly                                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  40:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         13 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  40:                        - - Trp Arg Arg Arg Arg Asn Arg Arg Pro Thr Se - #r Tyr Gly                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  41:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         7 amino acids                                      (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  41:                        - - Lys Ala Lys Ala Lys Ala Lys                                               1               5                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:  42:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         68 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "Lys" in position 66 has an                             n-X subst - #itution.                                            - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  42:                        - - Gln Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gl - #y Gly Glu Leu Val Asp        1               5  - #                10  - #                15                - - Thr Leu Gln Phe Val Cys Gly Asp Arg Gly Ph - #e Leu Phe Ser Arg Pro                   20      - #            25      - #            30                    - - Ala Ser Arg Val Ser Arg Arg Ser Arg Gly Il - #e Val Glu Glu Cys Cys               35          - #        40          - #        45                        - - Phe Arg Ser Cys Asp Leu Ala Leu Leu Glu Th - #r Tyr Cys Ala Thr Pro           50              - #    55              - #    60                            - - Ala Lys Ser Glu                                                           65                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:  43:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         68 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "Lys" in position 4 has an                              n-X subst - #itution.                                            - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  43:                        - - Gln Ala Tyr Lys Pro Ser Glu Thr Leu Cys Gl - #y Gly Glu Leu Val Asp        1               5  - #                10  - #                15                - - Thr Leu Gln Phe Val Cys Gly Asp Arg Gly Ph - #e Leu Phe Ser Arg Pro                   20      - #            25      - #            30                    - - Ala Ser Arg Val Ser Arg Arg Ser Arg Gly Il - #e Val Glu Glu Cys Cys               35          - #        40          - #        45                        - - Phe Arg Ser Cys Asp Leu Ala Leu Leu Glu Th - #r Tyr Cys Ala Thr Pro           50              - #    55              - #    60                            - - Ala Arg Ser Glu                                                           65                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:  44:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         38 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "Lys" in position 25 has an                             n-X subst - #itution.                                            - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  44:                        - - Tyr Ala Cys Asp Thr Ala Thr Cys Val Thr Hi - #s Arg Leu Ala Gly Leu        1               5  - #                10  - #                15               Leu Ser Arg Ser Gly Gly Val Val Lys Asn As - #n Phe Val Pro Thr Asn                        20      - #            25      - #            30                   Val Gly Ser Lys Ala Phe                                                                35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  45:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         35 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "Xaa" stands for an unnatural           amino                                                                                           acid with - # R group forming a ring                                           attached - #to "Asp" in position 1.                                            "Lys" - #in position 22 has an                                                 n-X subst - #itution.                                            - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  45:                        - - Asp Thr Ala Thr Xaa Tyr Thr His Arg Leu Al - #a Gly Leu Leu Ser         Arg                                                                               1               5  - #                10  - #                15               - - Ser Gly Gly Val Val Lys Asn Asn Phe Val Pr - #o Thr Asn Val Gly Ser                    20     - #             25     - #             30                   - - Lys Ala Phe                                                                       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:  46:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         68 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "Lys" in position 55 has an                             n-X subst - #itution.                                            - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  46:                        - - Gln Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gl - #y Gly Glu Leu Val Asp        1               5  - #                10  - #                15                - - Thr Leu Gln Phe Val Cys Gly Asp Arg Gly Ph - #e Leu Phe Ser Arg Pro                   20      - #            25      - #            30                    - - Ala Ser Arg Val Ser Arg Arg Ser Arg Gly Il - #e Val Glu Glu Cys Cys               35          - #        40          - #        45                        - - Phe Arg Ser Cys Asp Leu Lys Arg Leu Glu Th - #r Tyr Cys Ala Thr Pro           50              - #    55              - #    60                            - - Ala Arg Ser Glu                                                           65                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:  47:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         78 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  47:                        - - Asn Thr Leu Cys Gly Ala Glu Leu Val Asp Al - #a Leu Gln Phe Val Cys        1               5  - #                10  - #                15                - - Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Th - #r Gly Tyr Gly Ser Ser                   20      - #            25      - #            30                    - - Ser Arg Arg Ala Pro Gln Thr Gly Ile Val As - #p Glu Cys Cys Phe Arg               35          - #        40          - #        45                        - - Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cy - #s Ala Pro Leu Arg Pro           50              - #    55              - #    60                            - - Ala Arg Ser Ala Arg Ser Val Arg Ala Gln Ar - #g His Thr Asp               65                  - #70                  - #75                                - -  - - (2) INFORMATION FOR SEQ ID NO:  48:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         49 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "Lys" in position 1 has an                              n-X subst - #itution.                                            - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  48:                        - - Lys Gly Leu Pro Lys Glu Val Pro Ala Val Le - #u Thr Lys Gln Lys Leu        1               5  - #                10  - #                15                - - Lys Ser Glu Leu Val Ala Asn Gly Val Thr Le - #u Pro Ala Gly Glu Met                   20      - #            25      - #            30                    - - Arg Lys Asp Val Tyr Val Glu Leu Tyr Leu Gl - #n His Leu Thr Ala Leu               35          - #        40          - #        45                        - - His                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:  49:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         48 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "Lys" in position 4 has an                              n-X subst - #itution.                                            - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  49:                        - - Gly Leu Pro Lys Glu Val Pro Ala Val Leu Th - #r Lys Gln Lys Leu Lys        1               5  - #                10  - #                15                - - Ser Glu Leu Val Ala Asn Gly Val Thr Leu Pr - #o Ala Gly Glu Met Arg                   20      - #            25      - #            30                    - - Lys Asp Val Tyr Val Glu Leu Tyr Leu Gln Hi - #s Leu Thr Ala Leu His               35          - #        40          - #        45                        - -  - - (2) INFORMATION FOR SEQ ID NO:  50:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         697 amino acids                                    (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  50:                        - - Gln Arg Lys Arg Arg Asn Thr Ile His Glu Ph - #e Lys Lys Ser Ala Lys        1               5  - #                10  - #                15                - - Thr Thr Leu Ile Lys Ile Asp Pro Ala Leu Ly - #s Ile Lys Thr Lys Lys                   20      - #            25      - #            30                    - - Val Asn Thr Ala Asp Gln Cys Ala Asn Arg Cy - #s Thr Arg Asn Lys Gly               35          - #        40          - #        45                        - - Leu Pro Phe Thr Cys Lys Ala Phe Val Phe As - #p Lys Ala Arg Lys Gln           50              - #    55              - #    60                            - - Cys Leu Trp Phe Pro Phe Asn Ser Met Ser Se - #r Gly Val Lys Lys Glu       65                  - #70                  - #75                  - #80         - - Phe Gly His Glu Phe Asp Leu Tyr Glu Asn Ly - #s Asp Tyr Ile Arg Asn                       85  - #                90  - #                95                - - Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gl - #y Thr Val Ser Ile Thr                   100      - #           105      - #           110                   - - Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Se - #r Met Ile Pro His Glu               115          - #       120          - #       125                       - - His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Ly - #s Asp Leu Gln Glu Asn           130              - #   135              - #   140                           - - Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gl - #y Pro Trp Cys Phe Thr       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ser Asn Pro Glu Val Arg Tyr Glu Val Cys As - #p Ile Pro Gln Cys         Ser                                                                                              165  - #               170  - #               175              - - Glu Val Glu Cys Met Thr Cys Asn Gly Glu Se - #r Tyr Arg Gly Leu Met                   180      - #           185      - #           190                   - - Asp His Thr Glu Ser Gly Lys Ile Cys Gln Ar - #g Trp Asp His Gln Thr               195          - #       200          - #       205                       - - Pro His Arg His Lys Phe Leu Pro Glu Arg Ty - #r Pro Asp Lys Gly Phe           210              - #   215              - #   220                           - - Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gl - #n Pro Arg Pro Trp Cys       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Tyr Thr Leu Asp Pro His Thr Arg Trp Glu Ty - #r Cys Ala Ile Lys         Thr                                                                                              245  - #               250  - #               255              - - Cys Ala Asp Asn Thr Met Asn Asp Thr Asp Va - #l Pro Leu Glu Thr Thr                   260      - #           265      - #           270                   - - Glu Cys Ile Gln Gly Gln Gly Glu Gly Tyr Ar - #g Gly Thr Val Asn Thr               275          - #       280          - #       285                       - - Ile Trp Asn Gly Ile Pro Cys Gln Arg Trp As - #p Ser Gln Tyr Pro His           290              - #   295              - #   300                           - - Glu His Asp Met Thr Pro Glu Asn Phe Lys Cy - #s Lys Asp Leu Arg Glu       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Asn Tyr Cys Arg Asn Pro Asp Gly Ser Glu Se - #r Pro Trp Cys Phe         Thr                                                                                              325  - #               330  - #               335              - - Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Se - #r Gln Ile Pro Asn Cys                   340      - #           345      - #           350                   - - Asp Met Ser His Gly Gln Asp Cys Tyr Arg Gl - #y Asn Gly Lys Asn Tyr               355          - #       360          - #       365                       - - Met Gly Asn Leu Ser Gln Thr Arg Ser Gly Le - #u Thr Cys Ser Met Trp           370              - #   375              - #   380                           - - Asp Lys Asn Met Glu Asp Leu His Arg His Il - #e Phe Trp Glu Pro Asp       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Ala Ser Lys Leu Asn Glu Asn Tyr Cys Arg As - #n Pro Asp Asp Asp         Ala                                                                                              405  - #               410  - #               415              - - His Gly Pro Trp Cys Tyr Thr Gly Asn Pro Le - #u Ile Pro Trp Asp Tyr                   420      - #           425      - #           430                   - - Cys Pro Ile Ser Arg Cys Glu Gly Asp Thr Th - #r Pro Thr Ile Val Asn               435          - #       440          - #       445                       - - Leu Asp His Pro Val Ile Ser Cys Ala Lys Th - #r Lys Gln Leu Arg Val           450              - #   455              - #   460                           - - Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gl - #y Trp Met Val Ser Leu       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Arg Tyr Arg Asn Lys His Ile Cys Gly Gly Se - #r Leu Ile Lys Glu         Ser                                                                                              485  - #               490  - #               495              - - Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Se - #r Arg Asp Leu Lys Asp                   500      - #           505      - #           510                   - - Tyr Glu Ala Trp Leu Gly Ile His Asp Val Hi - #s Gly Arg Gly Asp Glu               515          - #       520          - #       525                       - - Lys Cys Lys Gln Val Leu Asn Val Ser Gln Le - #u Val Tyr Gly Pro Glu           530              - #   535              - #   540                           - - Gly Ser Asp Leu Val Leu Met Lys Leu Ala Ar - #g Pro Ala Val Leu Asp       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Asp Phe Val Ser Thr Ile Asp Leu Pro Asn Ty - #r Gly Cys Thr Ile         Pro                                                                                              565  - #               570  - #               575              - - Glu Lys Thr Ser Cys Ser Val Tyr Gly Trp Gl - #y Tyr Thr Gly Leu Ile                   580      - #           585      - #           590                   - - Asn Tyr Asp Gly Leu Leu Arg Val Ala His Le - #u Tyr Ile Met Gly Asn               595          - #       600          - #       605                       - - Glu Lys Cys Ser Gln His His Arg Gly Lys Va - #l Thr Leu Asn Glu Ser           610              - #   615              - #   620                           - - Glu Ile Cys Ala Gly Ala Glu Lys Ile Gly Se - #r Gly Pro Cys Glu Gly       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Asp Tyr Gly Gly Pro Leu Val Cys Glu Gln Hi - #s Lys Met Arg Met         Val                                                                                              645  - #               650  - #               655              - - Leu Gly Val Ile Val Pro Gly Arg Gly Cys Al - #a Ile Pro Asn Arg Pro                   660      - #           665      - #           670                   - - Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala Ly - #s Trp Ile His Lys Ile               675          - #       680          - #       685                       - - Ile Leu Thr Tyr Lys Val Pro Gln Ser                                           690              - #   695                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:  51:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         21 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  51:                        - - Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Cy - #s Val Tyr Phe Cys His        1               5  - #                10  - #                15                - - Leu Asp Ile Ile Trp                                                                   20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:  52:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         14 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  52:                        - - Asp Glu Glu Ala Val Tyr Phe Ala His Leu As - #p Ile Ile Trp                1               5  - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:  53:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         28 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  53:                        - - Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Ar - #g Met Asp Arg Ile Gly        1               5  - #                10  - #                15                - - Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Ar - #g Tyr                                   20      - #            25                                           - -  - - (2) INFORMATION FOR SEQ ID NO:  54:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         23 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  54:                        - - Gly Leu Phe Glu Ala Ile Ala Asp Phe Ile Gl - #u Asn Gly Trp Glu Gly        1               5  - #                10  - #                15                - - Met Ile Asp Gly Gly Gly Cys                                                           20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:  55:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         19 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  55:                        - - Lys Val Tyr Thr Gly Val Tyr Pro Phe Met Tr - #p Gly Gly Ala Tyr Cys        1               5  - #                10  - #                15                - - Phe Cys Asp                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:  56:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         23 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  56:                        - - Gly Gly Tyr Cys Leu Thr Arg Trp Met Leu Il - #e Glu Ala Glu Leu Lys        1               5  - #                10  - #                15                - - Cys Phe Gly Asn Thr Ala Val                                                           20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:  57:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         9 amino acids                                      (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  57:                        - - Glu Lys Gly Lys Gly Pro Gly Gly Lys                                        1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:  58:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         45 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  58:                        - - Tyr Lys Ala Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys        1               5  - #                10  - #                15                - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys                   20      - #            25      - #            30                    - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Trp Lys                   35                  - #40                  - #45                                - -  - - (2) INFORMATION FOR SEQ ID NO:  59:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         24 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  59:                        - - Cys Gly Leu Phe Glu Ala Ile Ala Asp Phe Il - #e Glu Asn Gly Trp Glu        1               5  - #                10  - #                15                - - Gly Met Ile Asp Gly Gly Gly Cys                                                       20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:  60:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         59 amino acids                                     (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  60:                        - - Gly Tyr Gly Pro Pro Lys Lys Lys Arg Lys Va - #l Glu Ala Pro Tyr Lys        1               5  - #                10  - #                15                - - Ala Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys                   20      - #            25      - #            30                    - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys               35          - #        40          - #        45                        - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Trp Ly - #s                               50              - #    55                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:  61:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         9 amino acids                                      (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  61:                        - - Tyr Lys Lys Ala Lys Ala Lys Ala Lys                                        1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:  62:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         100 amino acids                                    (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "Lys" in positions 3 to 100 may be                  present or absent.                                                   - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  62:                        - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys        1               5  - #                10  - #                15                - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys                   20      - #            25      - #            30                    - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys               35          - #        40          - #        45                        - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys           50              - #    55              - #    60                            - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys       65                  - #70                  - #75                  - #80         - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys                       85  - #                90  - #               95                 - - Lys Lys Lys Lys                                                                       100                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:  63:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         100 amino acids                                    (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "Arg Ala" in positions 3 to 100         may                                                                                            be present - #or absent.                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  63:                        - - Arg Ala Arg Ala Arg Ala Arg Ala Arg Ala Ar - #g Ala Arg Ala Arg         Ala                                                                               1               5  - #                10  - #                15               - - Arg Ala Arg Ala Arg Ala Arg Ala Arg Ala Ar - #g Ala Arg Ala Arg Ala                   20      - #            25      - #            30                    - - Arg Ala Arg Ala Arg Ala Arg Ala Arg Ala Ar - #g Ala Arg Ala Arg Ala               35          - #        40          - #        45                        - - Arg Ala Arg Ala Arg Ala Arg Ala Arg Ala Ar - #g Ala Arg Ala Arg Ala           50              - #    55              - #    60                            - - Arg Ala Arg Ala Arg Ala Arg Ala Arg Ala Ar - #g Ala Arg Ala Arg Ala       65                  - #70                  - #75                  - #80         - - Arg Ala Arg Ala Arg Ala Arg Ala Arg Ala Ar - #g Ala Arg Ala Arg Ala                       85  - #                90  - #               95                 - - Arg Ala Arg Ala                                                                       100                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:  64:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         100 amino acids                                    (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (ix) FEATURE:                                                                   (D) OTHER INFORMATION: - #  "Lys Ala" in positions 3 to 100         may                                                                                             be presen - #t or absent.                                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  64:                        - - Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala Ly - #s Ala Lys Ala Lys         Ala                                                                               1               5  - #                10  - #                15               - - Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala Ly - #s Ala Lys Ala Lys Ala                   20      - #            25      - #            30                    - - Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala Ly - #s Ala Lys Ala Lys Ala               35          - #        40          - #        45                        - - Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala Ly - #s Ala Lys Ala Lys Ala           50              - #    55              - #    60                            - - Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala Ly - #s Ala Lys Ala Lys Ala       65                  - #70                  - #75                  - #80         - - Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala Ly - #s Ala Lys Ala Lys Ala                       85  - #                90  - #               95                Lys Ala Lys Ala                                                                            100                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:  65:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:     - #         9 amino acids                                      (B) TYPE:     - #           amino acid                                         (C) STRANDEDNESS:   - #     single                                             (D) TOPOLOGY:    - #        linear                                    - -     (ii) MOLECULE TYPE:        - #    peptide                              - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  65:                        - - Lys Ala Lys Ala Lys Ala Lys Lys Tyr                                        1               5                                                            __________________________________________________________________________ 

We claim:
 1. Nucleic acid transporter system for delivering nucleic acid into a cell, comprising:a first nucleic acid binding complex comprising a first binding molecule noncovalently bound to said nucleic acid and covalently linked to a surface ligand; a second nucleic acid binding complex comprising a second binding molecule noncovalently bound to said nucleic acid and covalently linked to a nuclear ligand; wherein said first and second nucleic acid binding complexes are noncovalently bound to said nucleic acid simultaneously.
 2. The transporter of claim 1 further comprising a plurality of said first and second nucleic acid binding complexes.
 3. The transporter of claim 1 further comprising a third nucleic acid binding complex comprising a third binding molecule noncovalently bound to said nucleic acid and covalently linked to a lysis agent, wherein said first, second or third nucleic acid binding complexes are noncovalently bound to said nucleic acid simultaneously.
 4. The transporter of claim 3 further comprising a plurality of said first, second and third nucleic acid binding complexes.
 5. The transporter of claims 1 or 3 wherein said first and second binding molecules are selected from the group consisting of spermine, spermine derivative, spermidine, polylysine, histones, and cationic peptides.
 6. The transporter of claim 5, wherein said first and second binding molecules are a spermine derivative.
 7. The transporter of claims 1 or 3, wherein said surface ligand is a molecule which binds to a receptor selected from the group consisting of folate receptor, biotin receptor, lipoic acid receptor, low density lipoprotein receptor, asialoglycoprotein receptor, insulin-like growth factor type II/cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, or cartilage induction factor receptor.
 8. The transporter of claims 1 or 3, wherein said surface ligand includes a compound selected from the group consisting of Pep1 (SEQ ID NO:31), Pep2 (SEQ ID NO:32), Pep12 (SEQ ID NO:42), Pep13 (SEQ ID NO:43), Pep14 (SEQ ID NO:44), Pep15 (SEQ ID NO:45), Pep16 (SEQ ID NO:46), Pep17 (SEQ ID NO:47), Pep18 (SEQ ID NO:48), Pep19 (SEQ ID NO:49), Pep20 (SEQ ID NO:50), Pep21 (SEQ ID NO:51), Pep22 (SEQ ID NO:52), Pep23 (SEQ ID NO:53), A, B, G, D, E, P, J, M, asialoorsomucoid, folate, bone morphogenetic protein, cartilage induction factor, insulin-like growth factor-I or Fab'.
 9. The transporter of claim 3, wherein said nuclear ligand is a molecule selected from the group consisting of Pep3 (SEQ ID NO:33), Pep4 (SEQ ID NO:34), Pep5 (SEQ ID NO:35), Pep6 (SEQ ID NO:36), Pep7 (SEQ ID NO:37), Pep8 (SEQ ID NO:38), Pep9 (SEQ ID NO:39) and Pep10 (SEQ ID NO:40).
 10. The transporter of claims 1 or 3, wherein said surface ligand, nuclear ligand or lysis agent are attached to their respective binding molecule by a spacer.
 11. The transporter of claim 10, wherein said spacer is hydrophilic and has from 6-30 carbons.
 12. The transporter of claim 11, wherein said spacer has from 6-16 carbons.
 13. The transporter of claim 11, wherein said spacer is a repeating ω-amino acid of the structure

    [NH--(CH.sub.2 --CH.sub.2).sub.n --CO--].sub.m

wherein n=1-3 and m=1-20.
 14. The transporter of claim 11, wherein said spacer is a disulfide of the structure

    (CH.sub.2 CH.sub.2 --S--S--CH.sub.2 CH.sub.2 --).sub.n.


15. The transporter of claim 11, wherein said spacer is an acid sensitive bifunctional molecule of the structure ##STR99##
 16. The transporter of claim 3, wherein said lysis agent can be a virus, lytic peptide or lysis peptide.
 17. The transporter of claim 3, wherein said lysis agent is selected from the group consisting of adenovirus, parainfluenza virus, herpes virus, retrovirus, hepatitis virus, Pep24 (SEQ ID NO:54), Pep25 (SEQ ID NO:55), Pep26 (SEQ ID NO:56) or listeriolysin.
 18. The transporter of claims 1 or 3, wherein said second nucleic acid binding complex is selected from the group consisting of XXIIi, XXIIj, XXIIk, XXIIl, LIi, LIj, LIk, LIl, LXXIi, LXXIj, LXXIk, LXXIl, XCXi, XCXj, XCXk, XCXl, XXXi, XXXj, XXXk, XXXl, XXXIi, XXXIj, XXXIk, XXXIl, LXXIXi, LXXIXj, LXXIXk, LXXIXl, LXXXi, LXXXj, LXXXk, LXXXl, CVIIi, CVIIj, CVIIk, CVIIk, CVIIIi, CVIIIj, CVIIIk, CVIIIl, CXLIi, CXLIj, CXLIk, CXLIl, CXLIIi, CXLIIj, CXLIIk, CXLIIl and combinations thereof.
 19. The transporter of claims 1 or 3, wherein said first or second binding molecules are selected from the group consisting of IV, XXXIII, XI, XII, XL, XLI, LX, LXI, LXXXVIII, CX, XXIV, XXV, LXXIII, LXXIV, CII and combinations thereof.
 20. Nucleic acid transporter system for delivering nucleic acid into a cell, comprising: a first nucleic acid binding complex comprising a first binding molecule noncovalently bound to said nucleic acid and covalently linked to a surface ligand; a second nucleic acid binding complex comprising a second binding molecule noncovalently bound to said nucleic acid and covalently linked to a lysis agent;wherein said first and second nucleic acid binding complexes are noncovalently bound to said nucleic acid simultaneously.
 21. The transporter of claim 20 further comprising a plurality of said first and second nucleic acid binding complexes.
 22. The transporter of claim 20 wherein said first and second binding molecules are selected from the group consisting of spermine, spermine derivative, spermidine, polylysine, histones or cationic peptides.
 23. The transporter of claim 20, wherein the surface ligand is a molecule which binds to a receptor selected from the group consisting of folate receptor, biotin receptor, lipoic acid receptor, low density lipoprotein receptor, asialoglycoprotein receptor, insulin-like growth factor type II/cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetyl-choline receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor or cartilage induction factor receptor.
 24. The transporter of claim 20, the surface ligand includes a compound selected from the group consisting of Pep1 (SEQ ID NO:31), Pep2 (SEQ ID NO:32), Pep12 (SEQ ID NO:42), Pep13 (SEQ ID NO:43), Pep14 (SEQ ID NO:44), Pep15 (SEQ ID NO:45), Pep16 (SEQ ID NO:46), Pep17 (SEQ ID NO:47), Pep18 (SEQ ID NO:48), Pep19 (SEQ ID NO:49), Pep20 (SEQ ID NO:50), Pep21 (SEQ ID NO:51), Pep22 (SEQ ID NO:52), Pep23 (SEQ ID NO:53), A, B, G, D, E, P, J, M, asialoorsomucoid, folate, bone morphogenetic protein, cartilage induction factor, insulin-like growth factor-I or Fab'.
 25. The transporter of claim 20, wherein said lysis agent is a virus, lytic peptide or lysis peptide.
 26. The transporter of claim 20, wherein said lysis agent is selected from the group consisting of adenovirus, parainfluenza virus, herpes virus, retrovirus, hepatitis virus Pep24 (SEQ ID NO:54), Pep25 (SEQ ID NO:55), Pep26 (SEQ ID NO:56) or listeriolysin.
 27. The transporter of claim 20, wherein said surface ligand or said lysis agent is attached to the binding molecule by a spacer.
 28. The transporter of claim 27, wherein said spacer is hydrophilic and has from 6-30 carbons.
 29. The transporter of claim 28, wherein said spacer has from 6-16 carbons.
 30. The transporter of claim 28, wherein said spacer isa repeating ω-amino acids having a structure of

    [NH--(CH.sub.2 --CH.sub.2).sub.n --CO--].sub.m

where n=1-3 and m=1-20.
 31. The transporter of claim 28, wherein said spacer isa disulfide having the structure of

    (CH.sub.2 CH.sub.2 --S--S--CH.sub.2 --CH.sub.2 --).sub.n.


32. The transporter of claim 28, wherein said spacer is an acid sensitive bifunctional molecule of the structure ##STR100##
 33. The transporter of claims 1, 3, or 20, wherein said first nucleic acid binding complex is selected from the group consisting of Xa, Xb, Xc, XIa, XIb, XIc, XXXIXa, XXXIXb, XXXIXc, LIXa, LIXb, LIXc, LXXXVIIa, LXXXVIIb, LXXXVIIc, XIIId, XIIIe, XLIId, XLIIe, LXIId, LXIIe, XCId, XCIe, XVIIg, XLVIg, LXVIg, XCVg, XXIXg, LXXVIIIg, CVIg, XXVIIh, LXXVIh, CIVh, XIXh, XLVIIIh, LXVIIh, XCVIIh, CXLak, CXLbk, CXLck, CXLdk, CXLek, CXLgk, CXLhk, CXLfk, XXVId, XXVIe, LXXVd, LXXVe, CIIId, CIIIe, XXIIIa, XXIIIb, XXIIIc, LXXIIa, LXXIIb, LXXIIc, CIa, CIb, CIc and combinations thereof.
 34. The transporter of claims 3 or 20, wherein said second or third binding molecule covalently linked to said lysis agent are selected from the group consisting of XIIIf, XLIIf, LXIIf, XCIf, CXXIf, CXXVIf, XXVIf, LXXVf, CIIIf and combinations thereof.
 35. A DNA transporter system for inserting specific DNA into a cell, comprising, a plurality of a first DNA binding complex, said complex including a first binding molecule non-covalently binding to DNA, said first binding molecule covalently linked to a surface ligand, said ligand binding to a cell surface receptor;a plurality of a second DNA binding complex, said complex including a second binding molecule non-covalently binding to DNA, said second binding molecule is a cationic peptide and is covalently linked to a nuclear ligand, said nuclear ligand is a nuclear localization sequence that recognizes and transports the transporter system through a nuclear membrane; wherein the nuclear ligand is attached to the second binding molecule by a spacer, wherein said plurality of first and second DNA binding complexes simultaneously, non-covalently bind the specific DNA.
 36. The nucleic acid transporter system of claim 35, wherein said spacer is a hinge region of neutral amino acids.
 37. The nucleic acid transporter system of claim 36, wherein said hinge region is comprised of the amino acids glycine and serine.
 38. A DNA transporter for inserting specific DNA into a cell, comprising,a plurality of a first DNA binding complex, said complex including a first binding molecule non-covalently binding to DNA, said first binding molecule attached to a first spacer, said first spacer also attached to a surface ligand, said ligand binding to a cell surface receptor; a plurality of a second DNA binding complex, said complex including a second binding molecule non-covalently binding to DNA, said second binding molecule is a cationic peptide and is attached to a second spacer, said second spacer also attached to a nuclear ligand, said nuclear ligand is a nuclear localization sequence that recognizes and transports the transporter system through a nuclear membrane; a plurality of a third DNA binding complex, said complex including a third binding molecule non-covalently binding to DNA, said third binding molecule attached to a third spacer, said third spacer also attached to a virus; wherein said plurality of first, second and third DNA binding complexes simultaneously, non-covalently bind to the specific DNA.
 39. The nucleic acid transporter system of claim 38, wherein each spacer is a hinge region of neutral amino acids.
 40. The nucleic acid transporter of claim 39, wherein said hinge region is comprised of the amino acids glycine and serine.
 41. The transporter of any of claims 35 and 38, wherein the nuclear ligand is Pep3.
 42. The transporter of any of claims 35 and 38, wherein each said spacer is hydrophillic and has from 6-30 carbons.
 43. The transporter of claim 42, wherein each said spacer has from 6-16 carbons.
 44. The transporter of claim 43, wherein the spacer is a repeating omega-amino acid of the structure

    [NH--(CH.sub.2 --CH.sub.2).sub.n --CO--].sub.m

wherein n=1-3 and m=1-20.
 45. A method of introducing DNA into a cell comprising the step of contacting the cell with the transporter of any of claims 35 and
 38. 46. A method for in vivo targeting of the of the insertion of DNA into a cell comprising the step of contacting the cell with the DNA transporter of any of claims 35 and 38, wherein the surface receptor is specific to the cell which is targeted.
 47. The transporter of any of claims 35 and 38, wherein the plurality of second DNA binding complex are selected from the group consisting of XXIIj, XXIIk, XXIIl, LIi, LIj, LIk, LIl, LXXIi, LXXIj, LXXIk, LXXIl, XCXi, XCXj, XCXk, XCXl, of XXXi, XXXj, XXXk, XXXl, XXXIi, XXXIj, XXXIk, XXXIl, LXXIXi, LXXIXj, LXXIXk, LXXIXl, LXXXi, LXXXj, LXXXk, LXXXl, CVIIi, CVIIj, CVIIk, CVIIl, CVIIIi, CVIIIj, CVIIIk, CVIIIl, CXLIi, CXLIj, CXLIk, CXLIl, CXLIIi, CXLIIj, CXLIIk and CXLIIl.
 48. The transporter of of claim 47, wherein the plurality of second DNA binding complex are selected from the group consisting of CVIIi, CVIIj, CVIIk, CVIIl, and combinations thereof.
 49. The transporter of claim 47, wherein the plurality of second DNA binding complex are selected from the group consisting of XXXi, XXXj, XXXk, XXXl, and combinations thereof.
 50. The transporter of any of claims 35 and 38, wherein said nuclear ligand is selected from the group consisting of pep3, pep4, pep5, pep6, pep7, pep8, pep9, and pep10.
 51. A process for producing a transformed cell line, comprising the step of transfecting a cell with a nucleic acid transporter system according to any of claims 35 and 38, wherein said nucleic acid comprises a DNA sequence encoding a selectable marker.
 52. A process for producing a transformed cell line, comprising the step of transfecting a cell with a nucleic acid transporter system according to any of claims 35 and 38, wherein said nucleic acid encodes an oncogene. 